Ischaemia Flashcards
why do we need O2 for brain?
the immediate danger of hypoxia lies in the fact that oxygen is intimately coupled to the generation of ATP through OP, ATP needed for eg Na pump, the means to control volume, internal environment etc; Another problem with hypoxia is that a halt in OP means that the respiratory chain will stop pumping H+ out of the mitochondria. Thus, not only the cells but also their mitochondria may lose the membrane potential and become depolarized. This leads to the activation of apoptosis so cells may be doomed to die even if O2 suppl restored before necrosis begins; brain particularly vulnerable due to high energy use rate (ATP turnover 10x higher than average body tissue), ATP turned over every 5-10s and depleted if O2 deprived after a few secs; he brain is likely to be the first organ to lose energy charge and
depolarize when an animal is exposed to severe hypoxia or anoxia. This has two consequences. First, necrotic and apoptotic processes will be initiated rapidly in the brain of an animal that has lost its oxygen supply. Secondly, the depol brain can no longer regulate its volume, and the brain cells will start to swell. For many vertebrates, this is a particular problem because there is simply no space in the cranial cavity to allow the brain to swell. Therefore, instead of an increase in brain volume, there will be an increase in pressure in
the cranial cavity, and when this pressure rises above the blood pressure, it is no longer possible for blood to reach the brain. Even with the best health care, this is usually an irreversible situation, and consequently a lack of blood circulation
in the brain is a principal legal sign of death in many countries; fish/cold blooded animals often have much bigger cranial cavity than brain so it can swell 10%+ and be fine; humans, unconsciousness occurs and electric activity is suppressed in the brain just 6-7 s after blood flow to the brain is halted, likely to be an initial emergency response, possibly functioning to save energy by reducing ATPconsuming electrical activity. Moreover, as a standing person faints and falls, the brain moves into a lower position in relation to the heart, which increases cerebral blood pressure and thereby cerebral blood flow
selective neuronal loss
ischaemic infarct is as an area of pannecrosis, i.e., necrosis of all cell types including neurons, glia, and endothelial cells. The histologic appearance of the fully developed infarct is defined as coagulative necrosis. Injury to neurons in the rim surrounding the infarct has been termed general neuronal necrosis, since the other cell types are relatively intact. Some recent authors talk about this vulnerability as “selective neuronal loss”; where there is necrosis, there will also be inflammation, and this phenomenon may act
as a secondary insult
in SNL tissue bulk and extracellular matrix is preserved; SNL may be present in rescued penumbra after stroke, which hinders early clinical benefit and dampens long-term periinfarct plasticity; SNL can be hard to study, antibodies against neurons best as stains all meaning clear gaps where loss, but can stain acute neurons that are dead but not yet phagocytosed - nissl stains etc dont detect neurons that have been phagocytosed but stains neuropil and glia so hard to detect gaps; clinical correlates of SNL are difficult to address because in the vast majority of patients there is an associated infarct, albeit
small in most; some studies underlined their patients’ excellent-to-full neurological recovery despite sometimes extensive cortical SNL
impeding neuronal activation; plastic processes may develop over time so that the neural
functions impaired by SNL are taken over by neighboring neurons/areas, and second that standard clinical stroke scales may not capture subtle cognitive or sensorimotor impairments, as
suggested in recent studies after minor stroke or transient ischemic attacks
mechanism(s) underlying SNL in the salvaged penumbra, including why neurons die but other cells do not, are incompletely understood. Even the time course of SNL, i.e. when exactly the neurons die after reperfusion, is not well known; very high energy consumption of neurons makes them more sensitive to oxygen/glucose deprivation than other CNS cells, and hence more susceptible to ischemic injury during the occlusion phase; conversely, their very high postreperfusion mitochondrial activity would make them prone to
oxygen radical production and damage. Also, glutamate excitotoxicity or glutamate/GABA imbalance are also more likely to affect neurons than glial cells. One major hypothesis therefore is that SNL reflects irreversible cell damage ignited during the period of severe ischemia, which, if true, would imply that the degree of SNL should be proportional to both the severity and duration of
hypoperfusion. The major alternative hypothesis is that SNL is secondary to detrimental processes triggered by reperfusion—socalled ‘reperfusion injury’—such as oxygen radicals or inflammatory
processes, particularly microglia activation. A dual mechanism, requiring first the ischemic insult and then secondary reperfusion injury, may also be at play; inflammation also in SNL and maybe neurotoxic cytokines have a role
SNL likely reduces the ‘neuronal reserve’ that normally mitigates the motor, gait, and cognitive effects of age-related brain injury including small strokes, white matter damage, and neurodegenerative pathology. In this sense, among other established vascular factors, SNL following stroke and perhaps also transient ischemic attacks may contribute to poststroke cognitive decline; SNL may also occur in remote areas after focal lesion, esp thalamus and midbrain, with mechanism unknown but underpinned by combo or antero and retrograde degen; As EPND (Exofocal postischemic neuronal cell death) develops in a delayed manner, while patients are recovering from their deficits, and is uncoupled from the maturation of the primary ischemic lesion, it may be a target for (secondary) neuroprotection. At present, however, it is not entirely clear whether EPND has any functional/clinical relevance; it becomes detectable on MRI/histologically days or weeks after stroke; cortical granular atrophy or watershed territory microemboli in the cerebral cortex distal to large cerebral arterial occlusion in ICA or MCA due to athrosclerosis and SNL may occur but has not been demonstrated in humans yet
neuropath of globlal ischaemia
Reversible global ischaemia results in the death of certain selectively vulnerable, or regionally vulnerable neuronal populations. For example, pyramidal neurons in the CA1 subfield of the hippocampus will die after only 5 to 10 minutes of global ischaemia, while neurons in the nearby CA3 region remain intact; neurons selectively vulnerable to ischaemia include cortical pyramidal neurons (middle laminae), cerebellar Purkinje cells, hippocampal CA1 pyramidal neurons and subpopulations in the amygdala, striatum, thalamus and brain stem nuclei; depends on ischaemic sensitivity (cell type, brain region), delay interval (time after cerebral recirculation), time for irreversible injury to evolve (delayed neuron degen)
characteristics of selective neuronal necrosis (SNN) have been defined as a series of histologic
changes encompassed by the term ischaemic cell change; at least 3 time related properties of SNN: ischaemic interval required for initiating irreversible injury (ischaemic sensitivity), the time after cerebral
recirculation before irreversible injury begins (delay interval), and the time it takes for irreversible injury to evolve once it has been initiated; ischaemic sensitivity varies - neurons in CA1
succumb to periods of depolarising ischaemia lasting as little as 3 minutes, while periods of 15 to 20 minutes of severe ischaemia are needed to kill medium-sized spiny neurons in the striatum; the
interval between cerebral microcirculation and the initiation of irreversible injury differs markedly among neuronal populations. The comparatively ischaemia-resistant medium-sized striatal neurons show signs of irreversible injury within 3 hours of recirculation, while the onset of irreversible injury to the ischaemiasensitive CA1 neurons is apparently delayed by 48 to 72 hours; (smith et al with CAl/4 after 4 mins, CA3 after 6, and caudoputamen + middle laminae after 8-10)
hippocampal lesions in global ischaemia
Neurons in the central nervous system have different vulnerability to ischemic injury in a quite inhomogeneous way; some parts of HC very vulnerable meanwhile DG is close by and can tolerate considerable degree of ischaemia; In 1880, Sommer reported that one-third of epileptic patients showed extensive loss of neurons in a circumscribed portion of the hippocampus. He hypothesized that the lesion was due to repeated ischemia, provoked during the seizure attacks. This
area is now called Sommer’s sector, which corresponds approximately to the CA1 subfield; seems to explain secondary temporal lobe epilepsies in epilepsy patients; In Mongolian gerbils, bilateral carotid occlusion for 5 min always produced the hippocampal lesions
lactate transport and signalling in the brain
monocarboxylate transporters carry lactate, pyruvate, ketone bodies across brain cell membranes; this is facc diffusion; equilibrating action of MCTs also provides the basis for lactate acting as a volume transmitter that can mediate metabolic signals through the nervous tissue, as it can bind to the lactate receptor GPR81 on brain cells and cerebral blood vessels, resulting in inhibition of adenylyl cyclase; Lactate acts as a ‘buffer’ between glycolysis and oxidative
metabolism. In this process, it is exchanged as a fuel between cells and tissues with different glycolytic and oxidative rates; normally efflux from brain to blood but when blood lactate levels rise, such as in physical exercise, there is an influx of lactate from blood to brain; there is an instant decrease in lactate immediately after neuronal activation, followed after a few seconds by a rise, indicating that the cells have a latent capacity
for lactate oxidation that is rapidly exhausted; uptake cycle in which proton binds before the lactate anion, followed by a domain rearrangement during which lactate and proton pass through the channel between the extracellular and intracellular binding site to get released to the cytosol, lactate
first and then proton; lactate modifies prostaglandin action contributing to vasomotor
regulation, and influences the NADH/NAD+ ratio contributing to redox regulation; GPR81, a G-protein-coupled orphan receptor was previously
discovered to be selectively activated by lactate, downregulating cAMP through Gi; GPR81 is therefore subsequently named hydroxycarboxylic acid receptor 1 HCAR1; in adipose lactate serves autocrine role when adipocytes release high concs of lactate due to insulin dependent glucose uptake
MCT2 is concentrated at the postsynaptic membranes of glutamatergic synapses on spines of Purkinje cells in cerebellum and pyramidal cells in the hippocampus The density is lower at mossy fiber synapses, which have lower prevalent firing rate, compared with Schaffercollateral synapses. The lack of MCT2 at inhibitory type synapses on pyramidal cell somata suggests a connection to glutamatergic neurotransmission; role of lactate in synaptic plasticity is consistent with the observation that MCT2 expression is enhanced, together with postsynaptic density 95 protein and glutamate receptor subunit 2 (GluR2) protein, by BDNF, central mediator of synaptic plasticity; oligodendrocytes take up lactate and ketone bodies to synthesise myelin and after myelination has taken place, releases lactate to be used by axons
MCT1 knockout caused pathology similar to ALS and ALS patients show reduced MCT1 expression failure in the supply of lactate (generated in oligodendrocytes by glysolysis) could be involved in ALS and other degen diseases; lower cAMP levels may serve to regulate multiple cellular processes, including curbing glycolysis and glycogenolysis to save energy and limit acidification when glucose breakdown is faster than oxidation through mitochondria. This may counteract damage in hypoxic conditions,
but may even have a role in the first moments of cell activation, when glucose uptake and its conversion to lactate increase earlier than oxygen uptake; lowering cAMP using lactate could help preserve working memory (action of cAMP on HCN); Lactate stimulates production of BDNF by
astrocytes and neuronal cells, and BDNF helps regulate plasticity: possible mechanism for how exercise boosts the brain
pasteur effect and comparative anoxia tolerance
in anoxia inc’d need for anaerobic glycolysis which has thus inc’d requirement for glucose (but even maximal rate of glycolysis insufficient)
some indications that the marine mammalian brain has an enhanced glycolytic capacity. The glycogen content of the seal brain is some three times higher than that of the land mammal; Furthermore, 20-25% of the brain glucose uptake of the Weddell seal is released as lactate even when breathing air, compared to less than 10% for the human brain; Man will suffocate if breathing is stopped for more than 3-4 minutes; Weddell seals can remain submerged, actively swimming, for an hour or so; lutz et al 2004, brain of freshwater can withstand anoxia for days due to a fascinating, interlocking
cluster of adaptations that produce a state of deep
hypometabolism, the most effective of all hypoxia defense strategies; turtle brain is remarkable in having glycogen concentrations about fivefold greater than that of the rat which provides an immediately accessible store of glucose for anaerobic glycolysis until adequate glucose
supplies are liberated (recruited) from the large glycogen stores in the liver; Three distinct phases are involved in fully surviving anoxia:
(1) a drastic and immediate downregulation of ATP demand processes, (2) long-term maintenance at basal levels of ATP expenditure and (3) a rapid upregulation when oxygen becomes available; in mammlas NF-kB levels inc’d after stroke rapidly, turtle it translocates much slower as part of a preemptive defense mechanism against reoxygenation ROS damage; in the anoxic turtle brain gene transcription of Kv1 channels reduced to 18.5% of normoxic levels; almost full suppression in turtle brain electrical activity during anoxia; Neuronal network integrity is preserved through
the continued operation of core activities. These include periodic electrical activity, an increased release of GABA and a continued release of glutamate and dopamine
hypoxia and the brain
at PaO2 50 mm Hg there is an increase in the [lactate]/[pyruvate] ratio and a decrease in brain tissue pH. Increased lactate production is
accompanied by a rise in the cerebral metabolism of glucose, indicating accelerated glycolysis. The
cytosolic redox potential (measured by the NADH:NAD+ ratio) is shifted toward a more reduced state at PaO2 below 45 mm Hg. Phosphocreatine (PCr) concentration starts to decline and then fall precipitously below a PaO2 20 mm Hg. The steep fall in PCr coincides with
a decrease in ATP and reflects failure of mit respiration and OP; studies demonstrate that
cerebral energy metabolism remains normal when mild to moderate hypoxia (PaO2 = 25- 40 mm Hg) results in severe cognitive dysfunction in human volunteers. In mild hypoxia, CBF increases in order to maintain oxygen delivery to the brain. CBF can increase only about twofold; beyond this, the
cerebral metabolic rate for oxygen starts to fall and symptoms of hypoxia occur; In normal individuals exposed acutely to high altitudes there are the following problems. First, at altitudes of around 10,000 feet, as PaO2 falls below 45 mm Hg, difficulties with complex learning tasks and short-term memory appear. Second, above 20,000 feet, where PaO2 = 30mm Hg, cognitive disturbances and problems with motor co-ordination appear. Acute hypoxia of less than 20 mm Hg generally results in coma. Graded hypoxia in experimental animals reveals that brain ATP levels remain normal at PaO2 20 to 25 mm Hg, despite EEG slowing and a 50% reduction in PCr; so 50mmHg inc lac:pyru, inc glyco, no functional effects, 45mmHg inc CBF, inc redox state reduction, learning difficulty and memory loss, 30mmHg motor incoordination, 20mmHg PCR falls (starts falling from 40mmHg ish) but ATP preserved, coma
hypoxia inducible factor 1
Heterodimeric transcription factor alpha normally degraded (is hydroxylated at conserved proline residues allowing ubiquination then proteasome degradation, enzyme that adds OH needs O2 as substrate), stabilised due to hypoxia and binds to beta then translocates to nucleus; Adaptive response to hypoxia with time course 2 to 4 hours and induces glycolysis; HIF-1a accumulates in hypoxia; upregs genes involved in glycolsis, as well as VEGF (angiogenesis) by binding to HIF response elements
safeguarding against underperfusion
Cerebral perfusion is safeguarded through brainstem regulation of other circulations with first control of BP through neural regulation of CO and TPR, then when necessary the perfusion of
peripheral organs (except heart) is sacrificed; Baroreceptor unloading leads to tachycardia, increased contractility, vaso and venocontriction etc; intravascular injection to visualise vessels; striking density of intracortical vascular network; moody et al: few arteriole to arteriole anastomoses but there is a continuous cap network allowing weak anastomotic flow; A multiple supply is found in certain brain regions where adjacent arterioles arise from two or three widely separated surface (pial) arterial sources.
1nterdigitation describes an overlapping and interpenetration of adjacent arteriolar territories; the imaginary boundaries of perfusion responsibility in the capillary bed between these
arterioles fit together like a jigsaw puzzle rather than smoothly; last 2 occur rarely in human brains and seem to give inc’d protection; regions in the human cerebrum with interdigitating arterioles arising from different parent arteries on the surface
of the brain are the subcortical U fibers and the external capsule area. All other areas of
the cerebrum have arterioles from adjacent sources with capillary beds that each nourish a cylinder of brain, and other arterioles usually do not penetrate that particular unique cylinder
interdigitation of arterioles from separate sources offers favored regions of the brain unique protection from hypotensive events due to better collateral flow from caps; cortex and corpus callosum offered some protection from hypotension by the short distance from the pial plexus, and by having an afferent supply solely from arterioles (dont usually get atherosclerosis); centrum semiovale, basal ganglia, and thalamus are supplied by long arteries and arterioles and do not have interdigitating arteriolar fields. These regions are the most frequent sites for small ischemic infarcts, their arteries are subject to luminal narrowing from atherosclerosis (especially in hypertension and diabetes mellitus) and other degenerative vessel wall conditions; cortex is vulnerable to circulatory hypoperfusion in those areas in which distal middle and anterior (or posterior) cerebral artery distributions meet due to watershed infarct; cortex and BG/thalamus at risk of anoxia, centrum, BG, thalamus at risk of hypoperfusion (and cortex at watershed)
cerebral bloodflow autoreg
Over a normal range of blood pressure (mean between 50 and 140 mm Hg) CBF is kept constant by alteration in cerebrovascular resistance. Partial pressure of carbon dioxide and oxygen also influence CBF - increased by rising PaCO2 or falling PaO2 This effect is absent in hypotension; autoregulatory curve may be shifted to the right under conditions of increased sympathetic activity; upstream resistance vessels have increased tone, because these have sympathetic innervation, the effect is less capacity for downstream resistance vessels to increase flow by even maximal vasodilation (i.e., the socalled waterfall effect of two resistances in series)
capacity of an organ to regulate its blood supply in accordance with metabolic needs, or for brain: capacity of CBF to remain constant despite variation in BP; CBF first alters passively with BP
changes but then returns to initial values despite maintained change in BP (30s to 3 mins); Large cerebral arteries account for ~50% vascular resistance and receive PS (ACh, VIP) and symp (NA, NPY) innervation, as well as innervation from nociceptive c-fibres; the microcirculation contributes 50% of vascular resistance and don’t have symp reg but do account for CO2 (pH)
and O2 responsiveness; Lower limit of autoreg: Arteries and Arterioles dilate with falling CPP until Critical Pressure and Upper limit of Autoregulation
Arteries (> 400 µm), not Arterioles, constrict as CPP rises to Critical Pressure; thus autoreg works between these two limits
neurovasc coupling
state where regional CBF is increased to meet regional changes in brain tissue metabolism or activity. NVC consists of three brain cell types: neurons, supporting cells (astrocytes), and vascular cells (vascular smooth muscle, pericyte, and endothelial cells). These cells can be grouped into three conceptual components: neurons, the message senders associated with information processing; supporting cells, the potential transmission sites that mediate vasoactive signals
in response to the neuronal messages; and vascular cells, the recipients of the signals. After evoked neural activation, vasoactive signals are transmitted directly and indirectly via supporting cells to the vascular cells, which cause redistribution of the local CBF; substantial evidence for the role of astrocytes in NVC and functional hyperaemia (FH, increase in CBF with increased activity); 1) astrocytes must be activated in some way by neuronal signals that cause FH; 2) removing astrocytic signalling specifically in time and spatial location must perturb or abolish increased CBF by increased neural activity; 3) specifically activating astrocytic signals in the absence of neuronal activity should lead to FH. Of these, the first requirement has significant experimental support, but the last two have not been fully addressed
role of astrocytes in neurovasc coupling
signaling from neurons in activated brain regions
to local penetrating arterioles (and possibly also capillaries) and a coordinated response of surface vessels, are necessary for local CBF to increase during neuronal activation; changes in hemodynamics can appear within 1-3 s of increased neural activity, while metabolic changes occur more slowly than this (so not just CO2, O2 etc) and oxygen consumption occurs in
a much smaller area than the subsequent CBF increase; ionotropic glutamate receptor activation may mediate functional hyperemia by calcium-activated synthesis of nitric oxide (NO), prostaglandins etc in neurons; stimulation
of cortical astrocytes, either directly or through nearby neurons, triggers an intraastrocytic calcium surge and a subsequent dilation or constriction of neighboring arterioles. Vasodilation was triggered by activation of astrocytic metabotropic glutamate
receptors (mGluR) and either cyclooxygenase products or K channels on astrocytes and smooth muscles; astrocytes also release precursors for 20-HETE to make vasoconstriction, and cause changes in vascular tone by NO levels too; a study found astrocytic calcium elevations in
somatosensory cortex in awake mice, which appeared 1-2 s after the onset of voluntary running; astrocytic Ca++ changes observed after FH but maybe astrocytes maintain and eg NO triggers initial FH; ischaemia and global hypoxia reduce FH; CBF inc’s during spreading depol (migraine aura); Astrocytes swell under ischemia, and because of their proximity to arterioles
and capillaries, this edema may contribute to the CBF reduction in the microcirculation after stroke (but they are neuroprotective); also participate in
spreading neocortical depolarizations but unknown to what level they influence CBF here
glutamate and glut transporters
Glutamate and aspartate are non-essential amino acids that do not cross the blood-brain barrier. They are synthesised from glucose and a variety of other precursors within the brain. Synthetic and metabolic enzymes for glutamate and aspartate have been localised to neurons and glial cells. (Glutamic acid is in a metabolic pool with a-ketoglutaric acid and glutamine.) A large fraction of the glutamate released from nerve terminals probably is taken up into glial cells, where it is converted into glutamine. Glutamine then cycles back to nerve terminals, where it participates in the transmitter pools of glutamate and GABA; transmitter pool of glutamate is stored in synaptic vesicles that actively accumulate glutamate through a Mg2+/ATP-dependent process. Substances that destroy the electrochemical gradient inhibit this uptake mechanism. The concentration of glutamate within synaptic
vesicles is thought to be very high >20mM; glut/asp are zwitterions so cant cross PM; Uptake
mechanisms have an important role in regulating the extracellular concentrations of glutamate and aspartate in the brain. At least two families of
glutamate transporter have been localised to the plasma membrane of neurons and astrocytes. Only the Na+-dependent glutamate transporter is coupled to the electrochemical gradient that
permits transport of glutamate and aspartate against their concentration gradients; n the CNS, glutamate transporter-1 (GLT-1) and glutamate-aspartate transporter (GLAST) are expressed preferentially in glial cells, GLT-1 has its highest expression in the thalamus and cerebellum, with lower levels in the hippocampus, cortex and striatum. GLAST immunoreactivity is found
predominantly in the cerebellum and less so in the forebrain. Excitatory amino acid carrier-1 (EAAC1) is
expressed predominantly in neurons, most prominently in the hippocampus and not in glial cells; glut in brain tissue at 1-2mM
glut transporters major role limit glut/asp conc in ecf to prevent excessive GLUTr stim; Net transport of glutamate is increased by high intracellular K+; upon dissociation of glutamate and Na+ from the transport machinery, cytoplasmic K+ binds to
the protein to be recycled into the extracellular compartment - 3Na, 1H, 1 glut in and 1 K out; OH or HCO3 accompanies K out
glutamate neurotoxicity
first recognised as a neurotoxin in the late 1960s; Glutamate, and other amino acids, given to immature animals induced acute neurodegen in the areas of the brain not well protected by the blood-brain barrier - notably the hypothalamic arcuate nucleus. The mechanisms of neurodegen are divergent, and activation of all classes of ionotropic glutamate receptors has been implicated; Olney J. Brain lesions, obesity, and other disturbances in mice treated with
monosodium glutamate. Science 1969;164:719-21: In newborn mice subcutaneous injections of msg
induced acute neuronal necrosis in several regions of developing brain including the hypothalamus; treated mice became obese and females were infertile; MSG usually safe to eat, small numbers of people may have headaches, flushing, inc HR etc from glut (chinese restaurant syndrome - outdated term) but most people getting this will be experiencing nocebo
glut neurotox in disease states
Neurolathyrism is a spastic disorder occurring in East Africa and India. There is degeneration of
lower and upper motor neurons. The condition is associated with the dietary consumption of the
legume Latyrus sativus (grass/white/indian pea). This plant contains a toxin b-N-oxalyl-a,b-diaminopropionic acid; bridges et al 1989, compound is potent non-NMDAr agonist, inc KA, AP5 doesnt affect the compounds toxicity , thus non-NMDAr have role in excitotoxicity; the receptor it most binds to is AMPAr; moorhem et al 2011; susceptibility of motor neurons in this condition plus maybe ALS (sensory neurons spared) for excitotoxicity may in part relate to their relatively high expression of AMPA receptors, esp Ca++ permeable ones; beta odap also inhibs Na dep glut transport, increasing ecf levels further
Rasmussen’s encephalitis: Antibodies to the GluR3 receptor function as agonists and induce
seizure-like activity in rabbits. Anti-GluR3 receptor reactivity had been found in the sera of children
with a particularly severe form of epilepsy called Rasmussen’s encephalitis, as well as in people
with other forms of severe epilepsy; twyman 1997: studies demonstrate that antisera and purified IgG
antibodies to GluR3 directly activate a subpop of GluRs; receptor activation was blocked by the competitive antagonist CNQX, thus indicating that the antibody epitope interacted with the AMPA/KA binding or an agonist binding site of the receptor. Blockade of antibody activity by CNQX also suggests that the antibody activation of the receptor was not due nonspecific interactions with the receptor channel protein; Ig dosnt usually cross BBB in large amounts, possibility that focal disruption of the blood-brain barrier, perhaps following trauma or infection, in a region that
provides access of autoantibodies to the appropriate epitope on the target receptor would seem feasible. This scenario would account for the focal nature of the immune lesion and would also suggest that in other regions of the brain where the blood brain barrier is intact, particularly
the other brain hemisphere, autoantibody fails to gain access to the target antigen and is spared. Consequently, at least three independent events could be hypothesized to initiate and perpetuate the disease state: first, disruption of the blood brain barrier by trauma or possibly infection;
second, the presence or ability to produce the GluR3B or related autoantibodies; and third, the presence or display of GluR3 on the neuron in a form that can be accessed by the immune system. A “three-hit” model would also be consistent with the rare incidence of this disease
Anti-NMDA-receptor encephalitis is an acute form of encephalitis, potentially lethal but with high
probability for recovery, caused by autoimmune reaction against NR1- and NR2-subunits of the
glutamate NMDA receptor. Different descriptions and syndromal designations for this disease
existed in the medical literature before 2007, when the causal associations were established the
condition received its current name. The disease is associated with tumours, mostly teratomas of
the ovaries, and thus is considered a paraneoplastic syndrome. However, there are a substantial number of cases with no detectable cancerous tissue; Domoic acid poisoning s an acute form of hippocampal (CA3) toxicity that was
reported in an outbreak of elderly subjects exposed to shellfish, domoate is a kainate like agonist that algae makes and accumulates in shellfish, anchovies, and sardines though illnes in humans only seen from shellfish consumption; AMPA and KA activation damages HC and amyg with short term memory loss, possibly seizures and death, and motor weakness
processes following energy failure
anoxic insult for >few secs produces predictable anoxic depol: depletion of energy stores within
neurons and glia with a concomitant acidosis, and release of free radicals. Depletion of energy stores
affects cellular metabolism, energy dependent ionic pumps, and the ability of cells to maintain resting membrane potential; Within seconds of an
ischaemic insult, normal brain electrical activity
ceases due to the activation of membrane K+ channels and widespread neuronal hyperpol. The
hyperpol may be due to opening of K+
channels responding to acute changes in local
concentrations of ATP, H+ or Ca2+, or it may reflect
altered non-heme metalloprotein associated
with regulation of specific K+ channels. This response (presumably protective) fails, however, to preserve high-energy phosphate levels in tissue as concentrations of PCr and ATP fall within minutes after onset; fall in PO2 during ischaemia enhances lactic acid production as cells undergo a Pasteur shift from a dependence on aerobic metabolism to a dependence on glycolysis. The resulting lactic acidosis decreases the pH of the ischaemic tissue from the normal 7.3 to intra-ischaemic values ranging 6.8 to 6.2 depending, in part, on the pre-ischaemic quantities of glycogen and glucose available for conversion to lactic acid. In addition, efflux of K+ from depolarising neurons results in prolonged elevations in extracellular [K+] and
massive cellular depolarisation. Rapid inactivation of O2-sensitive K+ channels by decreased PO2, may represent one mechanism whereby neurons put a brake on this ongoing K+ efflux; intracellular Na+ and Ca2+ rise and intracellular Mg2+ falls
subsequent phases of anoxic depol
phase II: extracellular concentrations of many neurotransmitters are increased. Depol-induced entry of Ca2+ via voltage-sensitive Ca2+ channels stimulates release of vesicular neurotransmitter pools, including the excitatory amino acid (EAA) neurotransmitter glutamate. At the same time, Na+-dependent uptake of certain neurotransmitters, including glutamate, is impaired. Highcapacity uptake of glutamate by the glutamate transporter couples the uptake of one glutamate and two
Na+ with the export of one K+ and one HCO3
- (or OH-); discharge of ion gradients means driving force for glut uptake lost; n addition, glutamate uptake by the widely expressed astrocyte high-affinity glutamate transporter (GLT-1), or EAA transporter-2 (EAAT2), and the
neuronal transporter EAAT3, can be down-regulated by free radical-mediated oxidation of a redox site on the transporter. Furthermore, since the transporter is electrogenic (i.e., normally transferring a positive charge inward), membrane depolarisation can lead to reversal of the transporter, producing glutamate efflux. Thus, both impaired glutamate uptake and enhanced glutamate release contribute to sustained
elevations of extracellular glutamate in the ischaemic brain. Microdialysis of ischaemic rat brain has detected an increase from the resting extracellular glutamate concentrations of 1 to 2 µM up to concentrations in the high µM to low mM range. However, not all of this is from the neurotransmitter pool: likely that most comes from the metabolic pool
mechanisms of glut release in brain ischaemia
Four release mechanisms have been postulated: vesicular release dependent on external calcium2 or Ca2+ released from intracellular stores; release through swelling-activated anion channels; an indomethacin-sensitive process in astrocytes;
and reversed operation of glutamate transporters. Here we have mimicked severe ischaemia in hippocampal slices and monitored glutamate release as a receptor-gated current in the CA1 pyramidal cells that are killed preferentially in ischaemic hippocampus. Using blockers of the different release mechanisms, we demonstrate that glutamate release is largely by reversed operation of neuronal glutamate transporters, and that it plays a key role in generating the anoxic depol that abolishes information processing in the central nervous system a few minutes after the start of ischaemia; most ischaemia current from glut release not K influx as higher [k]o only produced small inward current reduced by AP5 whereas applying glut gave large transient inwards current; ATP/GTP levels probably fall too low for much vesicular release so Ca++ unlikely to have big role, shown by replacing Ca++ with Mg++ in solution and adding Ca++ chelater; astrocyte swelling can activate channels leading to glut release but blocking this process didnt have sig effect on anoxic depol current; glut hom fails dramatically and transporters are the major source of the extracellular glutamate that triggers the death of neurons; modelling supported this conclusion
pannexin hemichannels in anoxic depol of hippocampal pyramidal cells
notion that this inward current reflects ion entry through glutamate-gated channels has been challenged by the discovery that, in isolated CA1 pyramidal cells, ischaemia evokes a large inward current even in the presence of glutamate receptor blockers; This current is inhibited by carbenoxolone and lanthanum (La3+) (Thompson et al., 2006), which block gap junction hemichannels. Hemichannels are made of connexin or pannexin proteins, they function as ion channels in neuronal and glial membranes and they are expressed at the post-synaptic densities of hippocampal pyramidal cells; activation of NMDA receptors is also reported to evoke a
current component mediated by hemichannels, which is blocked by carbenoxolone and by 10PanX (Thompson et al., 2008), i.e. blockers of hemichannels formed by pannexin-1, Thus, in ischaemia, pannexins might generate a large inward current, producing the anoxic depol, either because of a direct activating effect of ischaemia on pannexin hemichannels as seen in isolated pyramidal cells), or as a consequence of secondary hemichannel opening produced by ischaemia-evoked glutamate release activating NMDA receptors; used HC slice technique; in their expt, pannexin blockers didnt affect anoxic depol, and when glut-r blockers applied didnt block the slow inwards current that remained; Thus, pannexin-1 hemichannels do not contribute to the small and slow inward current, ruling out the
possibility that, when glutamate receptors are blocked, there is a significant activation of pannexin-1 hemichannels in the first 25 min of ischaemia
by varying ATP conc found magnitude and time course of the anoxic depolarization current do not depend on the availability of intracellular ATP and, whether or not ATP is present, they are not affected by hemichannel blockers; brain slices and therefore we cannot rule out the possibility that pannexins may have some effect on the response to stroke in vivo - eg maybe they have role in penumbra; Neuronal gap junctional hemichannels, composed of pannexin-1 subunits, have been suggested to play a crucial role in epilepsy
and brain ischaemia. After a few minutes of anoxia or ischaemia, neurons in brain slices show a rapid depolarization to 20 mV, called the anoxic depolarization. Glutamate receptor blockers can prevent the anoxic depolarization, suggesting
that it is produced by a cation influx through glutamate-gated channels. However, in isolated hippocampal pyramidal cells, simulated ischaemia evokes a large inward current and an increase in permeability to large molecules, mediated by the opening of pannexin-1 hemichannels. N-methyl-D-aspartate is also reported to open these hemichannels, suggesting that the activation
of N-methyl-D-aspartate receptors, which occurs when glutamate is released in ischaemia, might cause the anoxic depolarization by evoking a secondary ion flux through pannexin-1 hemichannels. We tested the contribution of pannexin hemichannels to the anoxic depol in CA1 pyramidal cells in the more physiological environment of hippocampal slices. Three independent inhibitors of hemichannels—carbenoxolone, lanthanum and mefloquine—had no significant effect on the current generating the
anoxic depolarization, while a cocktail of glutamate and gamma-aminobutyric acid class A receptor blockers abolished it. We conclude that pannexin hemichannels do not generate the large inward current that underlies the anoxic depol.
Glutamate receptor channels remain the main candidate for generating the large inward current that produces the anoxic depolarization
blood flow alteration in neuroprotection following ischaemia
During acute ischaemic stroke, the early restoration of oxygen and glucose to the ischaemic region is the best ‘neuroprotective therapy’. This is currently provided clinically by thrombolysis; Ischaemia has profound effects on CBF levels. Antegrade blood flow ceases during arterial occlusion, but collateral vessels may sustain cerebral perfusion in the arterial
bed, Ischaemic damage occurs when
collaterals fail to provide adequate perfusion leading to symptom onset; autoreg lost so mABP has large effect: inducing hypertension improves CBF and reduces injury after mca occlusion; upon reperfusion, significant hyperaemia within the ischaemic region immediately occurs but this is
followed by a post-ischaemic hypoperfusion which can last for hours. This is described as the ‘no-reflow phenomenon’ and has been attributed to the narrowing of capillaries and loss of both arteriolar dilating mechanisms and cerebrovascular
reactivity. Pericytes are susceptible to ischaemic injury resulting in contraction of capillaries
causing attenuated CBF, even after reperfusion; also reperfusion injury; for the nagel study, Laser Doppler flowmetry was used to measure relative CBF over the right somatosensory cortex of a male Wistar rat. Baseline CBF was normalized to 100% blood flow units (BFU). Upon temporary common carotid artery (CCA) ligation, CBF was reduced to 60% BFU; this went to 20% when MCA occluded, then hyperaemia to 80% followed by hypoperfusion of 40% seen
summary of energy failure and anoxic depol
What is the relationship between ischaemia- and
glutamate receptor-mediated neurotoxicity?; energy failure: Phase 1 Lengthen - KATP or hypothermia Phase 2 Slowed with hyperglycaemia or temperature Feature: voltage-sensitive Ca2+-
channels Phase 3 Features: energy failure, High [K+]o; how these contribute and interact: Depolarisation *Vesicular Glutamate release; Anoxia *Oxygen-sensitive metabolism blocked so Glutamine to Glutamate; High [K+]o *Remove Mg2+ block of NMDA receptor *Reversed uptake of Glutamate
depolarization process is energy-linked (ATP level); The link to ATP levels involves functioning of the Na+/K+-ATPase (inhibition under normoxia results in rapid depolarization similar to AD; Complete failure of Na+/K+-ATPase cannot account for the rate of change in extracellular ion concentrations. A change in membrane ion permeability must also occur; Blockade of GLUT-receptors alone or use of tetrodotoxin (Na+-channel) does not prevent AD; Low PO2 can directly affect the activity of membrane ion channels, but Pannexin channels do not contribute significantly to AD; AD appears to be a Ca2+-dependent process
In brain in absence of O2, glutamine degrades to glut and then to GABA; So eg CA1 vulnerability could be due to high NMDA conc (due to memory/learning); MK-801 is site for Mg++ binding where antags can bind (noncompetitive) or glut binding site (competitive); Glut is denditotoxic (blebbing seen on dendrites not soma); Toxicity matches receptor distribution (CA3 knocked out and CA1 spared by kainate, NMDA agonists spare CA3, take out CA1 and DG)
features of focal ischaemia
rim of the ischaemic core is recruited in to the
final infarct - how and why does this occur? In other words, what does/could the ischaemic penumbra tell us about focal ischaemia?; pharmacological approach was used to dissect
potential mechanisms. Consider the results of the
‘neuroprotection’ studies in a model of focal
ischaemia where infarct volume is used as the
experimental endpoint; NBQX gave no protection, MK801 did when given up to 8hrs after lesion; In focal ischaemia infarct volume is reduced when MK-801 is given to the animal, even as late as 8 hours after vessel occlusion. There is no protection with NBQX. What accounts for this time course and what does it tell us about the underlying mechanism of neurotoxicity? The most parsimonious hypothesis is that, after vessel occlusion in focal ischaemia, an NMDA-receptor-mediated (and not non-NMDA receptor mediated) form of neurotoxicity is on-going for some 8 hours. This phenomenon turned out to be cortical spreading depression; normal brain pH 7.2
is their sustained depol allowing mk-801 binding (by mg++ removal) for 8 hrs post lesion - is neuronal death delayed or is this secondary insult - the latter, and the insult is CSD; in penumbra there is increased glucose utilisation and O2 extraction
cortical spreading depression
SD is a transient depolarisation of the neuropil, which slowly spreads over the entire hemisphere, that can be triggered by a variety of pathologic events. When SD is propagated through normal cerebral cortex it does not produce major histopathological changes. SD is propagated by the release of glutamate, and the ionic changes including Ca2+ influx are similar to those evoked by anoxicischaemic depolarisations. Ion homeostasis
after SD is rapidly restored by marked activation
of ion transport that, in turn, is coupled to an
increase of metabolic activity and blood flow; SD-like depolarisation also occurs in the penumbra of focal ischaemia but the pathologic significance is
different from that in the intact brain. They come and go at irregular rates between 1 and 10 per hour, and they last much longer than the events in intact-non-injured brain. In focal ischaemia there is an absence of an appropriate metabolic-haemodynamic coupling (neurovascular coupling from earlier); increased oxygen requirements are not coupled to an increase in blood flow and, therefore, result in transient episodes of tissue hypoxia; the [k]o and Em of AD and SD differ, with AD more prolonged
how delayed CSD related to infarct growth
SpD may be triggered by high [K ]o and glutamate in the ischemic core and actively propagates tissue depolarization, ionic imbalances, and glutamate release into adjacent tissue. In the injury penumbra, where blood supply is compromised, SpD waves cause further reduction in tissue PO2 and exacerbate metabolic stress-failure, also called peri-infarct depols in thiz context; PIDs in the initial hours after injury have been shown to cause a step-wise increase in the ischemic tissue volume; MCAo filament occlusion (so genuine ischaemia induced); PIDs began soon after MCAo and recurred periodically over the subsequent 2 hr. After reperfusion, activity ceased and did not recur for the following 6 hr. At ~8 hr after injury, however, PID activity re-emerged in a delayed secondary phase. During this phase, which lasts 12 hr,PIDs recurred continuously and often in rapid succession; To explore the timing of infarct growth
relative to the secondary phase of PIDs, a
separate group of animals was killed at different postinjury times, and infarct areas of coronal sections were quantified; Rates of infarct growth at the caudal level changed with a time course nearly identical to that for PID frequency; NMDA receptor-mediated currents are involved in the
propagation of SpD/PID, and their antagonism has been shown to reduce PID occurrence; con-g (NMDAr antag) group had a significantly reduced incidence of PIDs associated with a 37% decrease in core infarct volume at 24 hr; The relative latency of DC deflections on electrodes over frontal and parietal regions varied for different PID events, implicating more than a single source for their initiation; First, latencies in the secondary phase were initially quite short, indicating more rapid
propagation speed, but grew progressively longer through subsequent events. Second, latencies were not randomly distributed but instead clustered around particular values. Third, the sequential order in which latency values occurred was also nonrandom. Ordered sequences included series of consecutive latencies with similar values or alternations between two values; topographic maps were constructed from multichannel recordings obtained in four animals
The most common PID pattern observed was a wave of negative DC deflections initiating in frontal regions and propagating caudally, PID waves also initiated in parieto-occipital regions and propagated to frontal areas, yielding negative latencies; PID early phase lower incidence in previous studies is likely attributable to the continuous use of anesthesia, which is neuroprotective and slows brain metabolism, and particularly halothane, which
reduces SpD/PID frequencies; However, as is the case with previous studies on PIDs during the ischemic phase, it is unknown whether reducing PID occurrences provides neuroprotection, or vice versa; e multiple foci for PID development
may reflect different mechanisms of initiation. Nedergaard and Hansen (1993) showed that the initiation of depolarization waves in MCAo can occur with characteristics of either SpD, as provoked by high [K ]o and glutamate, or ischemic depolarization resulting from hypoperfusion
in ischemic border zones. These processes
may occur in different cortical regions
Additionally, rostrocaudal waves may initiate in the striatum and propagate to the cerebral cortex, rather than initiate within the cortex itself. The rat brain is unique in that the claustrum and nucleus accumbens provide a rostral bridge of nearly continuous gray matter between the striatum and neocortex, and Vinogradova et al. (1991) demonstrated that SpD, occurring in the striatum, can traverse this bridge. Thus, the striatal infarct core might serve as an independent source of PIDs, which propagate to the cortex and contribute to its worsening pathology. This
additional source may, in part, account for the high incidence of PIDs in MCAo of the rat; in penumbra, PID metabolic challenge depletes ATP, induces permanent cell swelling, and reduces local blood flow; PID may be major form of damage in penumbra with glut release/excitotoxicity a secondary consequence: has been argued that the neuroprotective effects of glutamate receptor antagonists in brain injury models do not necessarily imply the occurrence of excitotoxic processes. In reducing PID frequency, admin of glutamate receptor antagonists reduces both infarct volume and ATP depletion (glut receptors needed for PID to spread); SPD-like events have been demonstrated in vivo in humans eg fMRI of migraine blood flow, application of KCl, although direct evidence is missing (as of 2003); PID inhib may be important part of neuroprotection; then fabricius et al 2005 provided direct electrophysiological evidence for the existence of PIDs and hence a penumbra in the human brain, by recording and finding SD post-acute brain injury (though not in everyone); late onset of PID phase 2 gives smaller infarct
what drives CSD in vivo - neurons or astrocytes - and in what ways is it similar to anoxic depol?
Neurons and their dendrites are the main active players of cortical SD. The depolarization is observed in the extracellular space as a prominent negative change of the slow potential. This slow potential change may result from longitudinal gradients of depolarization along neurons, probably owing to zonal dendritic opening of ion channels, which allow for large sustained influxes of small cations (sodium and calcium) in the neurons. Propagation is not the essential feature of cortical SD as the core process can be modeled in
a single neuron; During these electrophysiological transients there is virtual disappearance of pial arteries on the surface of the brain. The spreading depolarization precedes this deficit in perfusion
role of csd in spreading ischaemia
Spreading depolarization is characterized by near-complete breakdown of ion gradients, near-complete sustained depolarization in individual recordings of neurons, extreme shunt of neuronal membrane resistance, loss of electrical activity and neuronal swelling and distortion of dendritic spines. Thus, spreading depolarization represents an electrical change with near-complete short circuit between neurons and extracellular space, and a biochemical and morphological
alteration; Neurons cannot fire action potentials, as the sustained depolarization is above the threshold, thus causes electrical silence; possible that direct gap-junctional neuronto-neuron communication brings cells into synchronous operation and provides a transcellular pathway for the propagation of spreading depolarization where firing of neurons is not required. Moreover,
propagation can occur independently of astrocytic function; mutation in a CaV channel gene can reduce CSD as can loss of function of gene encoding astrocyte Na pump (prob due to inc glut transmission); normally in neurovascular coupling, ynaptic transmission are involved in the neurovascular response. Neuronal activation causes glutamate-evoked calcium influx in postsynaptic neurons that activates production of nitric oxide (NO) and arachidonic acid metabolites. Subsequent vasodilatation reflects both presynaptic activity and the level of postsynaptic depolarization and astrocytes sense glutamatergic transmission via metabotropic glutamate receptors and can signal vascular smooth muscle via their endfeet through arachidonic acid pathways; normally spreading depolarization induces an rCBF rise by more than 100%, which is called spreading hyperemia
artificially triggering SDs outside penumbra and propogating into it enlarged necrotic core (important evidence otherwise PID and core volume link may not be causation) (takano 1996); Inverse hemodynamic response is a marked, prolonged hypoperfusion due to severe arteriolar constriction that is coupled to spreading depol under pathological conditions; subarachnoid H+ can induce SD (and hence in the long paper in the flashcard above this one they discounted rats which had it, to avoid another source); spreading depol would normally cause vasodilation, but vasoconstriction instead as K is vasoconstrictor above certain conc, and NO synthesis is decreased - NO donors revert spreading ischaemia to normal spreading depression; two-dimensional imaging of rCBF showed that the region subject to severe ischemia enlarged with
each spreading depolarization due to inverse flow coupling(ask tasker about this); long lasting slow potential change (ecf index of SD) - prolonged SPC, indicating prolonged neuronal depol, results from mismatch between energy demand and supply, which causes insufficiency of membrane pumps to repolarize the neurons; negatively charged proteins inside the neuron cause small cations such as sodium and calcium to enter from
the extracellular space, producing a small dendritic inward current. Compensation for this constant inward current by dendritic outward current of the sodium pump establishes the so-called double Gibbs-Donnan equilibrium of the
physiological ion distribution, characterized by iso-osmolality across the membrane and steep physiological ion gradients. Bottom, the core
process of spreading depolarization is failure of sodium and calcium pumps to provide sufficient dendritic outward currents to balance the
persistent inward currents through pink and purple channels. If net dendritic current turns inward (persistent influx of sodium and calcium is more than the outflux of potassium), the double is shifted toward an almost simple Gibbs-Donnan equilibrium, characterized by near-complete
loss of electrochemical energy, almost passive ion distribution across the membrane, intracellular hyperosmolality with cellular swelling and
distortion of dendritic spines; and sustained depol to -10mV
neurovasc unit in csd
chuquet et al demonstrated that cortical SD
dramatically affects blood flow deep in the intact cortex; authors demonstrated two types of astrocytic [Ca2+] waves, one of which was
associated with haemodynamic change. A neuronal [Ca2+] wave of cortical SD generally
precedes the astrocytic [Ca2+] wave, and that interference with the latter prevents
vasoconstriction but not propagation of cortical SD. The vasoconstriction of intracortical
arterioles severe enough to result in arrest of capillary perfusion is correlated with fast
astrocytic [Ca2+] waves and is inhibited when they are reduced. Similar changes in [Ca2+] occur in AD, supporting the notion that AD and cortical SD are related - however the morphology of the
electrophysiology transient tells us that different ion channels must be involved; Pial vessels show
biphasic response but cortical vessels show
constrictor response
consequences of csd in penumbra
breakdown of ion homeostasis that can be recovered by ion pumps if the energy supply is adequate. In vivo, neurons in the penumbra area show rapid (<6 s), reversible dendritic beading; Dendrites quickly (<3 min) recover between waves of cortical SD to near-normal morphology until the occurrence of cortical SD-induced terminal dendritic injury occurs, signifying acute synaptic damage. The current hypothesis for the mechanism of expediting recruitment of the penumbra into the core zone of focal ischaemia is that metabolic stress, resulting from recurring waves of cortical SD, facilitates acute injury at the level of dendrites and dendritic spines in metabolically compromised tissue. These events have been observed in a number of species, including man; Last, despite their apparent detrimental role in infarct growth, it is possible that waves of cortical SD around a lesion might also initiate upregulation of neurobiological responses involved in repair and
remodeling. The above hypothesis is not new. It is a renewal of the so-called ‘weak excitotoxic hypothesis’ (i.e., neurons can be rendered susceptible to excitatory aminoacid neurotoxicity by manipulations of cellular energy metabolism and membrane potential). Numerous mechanisms have the potential to contribute to neurotoxicity, including impaired cytoplasmic Ca2+ homeostasis and release of oxygen free radicals, which exacerbate damage eg: Depressed Na extrusion from cytosol due to depressed Na/K-ATPase activity leads to Decreased Na/Ca exchange
Decreased Na-dependent glutamate uptake leads to Increased cytosolic Ca Increased extracellular
glutamate; Mitochondrial metabolic dysfunction leads to Mitochondrial depolarisation due to reduced proton extrusion from electron transport chain leads to Depressed Ca uptake by
mitochondria leading to increased cytosolic Ca; Mitochondrial metabolic dysfunction leads to
Increased mitochondrial production of ROS leads to Activation of the mitochondrial permeability transition pore Further metabolic impairment
In focal stroke, failure of the Na -K pump caused by depletion of ATP results in a wave of a cortical spreading depolarization (SD) of neurons and glia referred to as anoxic depolarization (AD) AD propagates through the stroke focus leading to dramatic neuronal and glial swelling, dendritic
beading, and spine loss; Without immediate reperfusion, AD spread defines the primary region of acute neuronal death, the ischemic core; As AD spreads throughout metabolically compromised penumbra surrounding the ischemic core, it becomes transient depolarization, often termed periinfarct depolarization (PID); proposed that each PID is initiated at the edge of ischemic core by elevated [K]o and glutamate; increased energy needs for recovery of ion gradients by ion pumps; Accompanying arteriole/capillary activity also contributes to the metabolic stress. Perfusion may decrease transiently during an SD in the penumbra,
resulting in abrupt reduction of cerebral blood flow; model of penumbra in conjunction with in vivo two-photon laser scanning microscopy to reveal dramatic spatiotemporal changes in dendritic integrity with the passage of each SD; intact control dendrite was beaded when imaged immediately after the initial induced SD at 8 min. After passage of the SD, the beading subsided; with passage of multiple SDs, dendrites undergo similar rounds of rapid beading and recovery
without a progressive increase in visible
structural damage until terminal beading
occurs; Rapid dendritic recovery occurred more frequently when there was a nearby flowing vessel (provide energy to restore); energy deprivation alters the conformation and distribution of the polymeric F-actin which contributes to the beading as well as water influx;the presence of a nearby flowing vessel was not a definitive indication of recovery since some beaded dendrites did not recover even when a flowing vessel was <20 microm away, so even residual flow doesnt stop incorporation into infarct by elevated metabolic stress
all waves (indicated by CBF) propagated circumferentially along the border of the ischaemic focus, including those arising outside the image field. They either cycled around the focus, often multiple times or they split at the point of origin and travelled then as two waves around either side of the focus until they met at the opposite side and annihilated each other; examination in inner zones near the ischaemic core showed biphasic hypo/hyperaemic or monophasic hypoperfusion CBF responses (that were sometimes sustained), whereas analysis of regions of interest in the outer zones always revealed monophasic hyperaemic responses; Thus, depolarization-coupled CBFIND responses in rat dMCAO are compatible with a gradient of perfusion developing in border zones of ischaemic foci after arterial occlusion, as described originally, to our knowledge, by Symon; seemed that events spreading radially most often
occurred early in the course of post-MCA occlusion observation, whereas circumferential spread was seen later; however, this could not be verified formally; principal finding in this study was the detection of a wave of cortical blood flow alteration secondary to a depolarization that
cycled, spontaneously and several times, around a focal ischaemic lesion in the lissencephalic brain of rats. This gave rise to the observation, at any single given point in the wave path, of recurrent blood flow transients with quite regular periodicity—in temporal terms a ‘cluster’—and the imaging data allowed not only interpretation of such clustering as indicating repetitive peri-lesion cycling of a single depolarization wave, but also demonstrated
considerable stepwise enlargement of the ischaemic lesion with each single wave cycle; demonstration of repetitive peri-lesion cycling of a
depolarization introduces the novel hypothesis that this behaviour may not simply cause lesion growth but also serve as a biological amplifier, enhancing upregulation around a lesion of inflammatory, stress- and neurogenic-response cascades to focal brain injury that are known to be related to spreading depolarization
peri-infarct hot zones
triggering factors for PIDs unknown; spontaneous origin in penumbra seemingly random; In peri-infarct tissue, transmembrane ionic gradients are preserved, but critically reduced cerebral blood flow (CBF) and oxygenation and mildly elevated extracellular potassium concentrations render the tissue highly susceptible to anoxic depolarization. Such conditions predict metastable hot zones, tenuously maintaining their membrane potential at steady state ischemia. To explain the origins of PID occurrence, we hypothesized that any sudden
worsening in metabolic supply-demand mismatch may transiently tip the balance towards anoxic depolarization in these hot zones, triggering a PID; tactile somatosensory stimulation during stroke significantly increases the rate of PID occurrence temporally coupled to the stimulus. The data also
suggested that PIDs are not triggered simply by a generalized arousal response to light tactile stimulation, because stimulation of the ipsilesional side to activate the non-ischemic hemisphere did not trigger PIDs more than what would be expected from spontaneously occurring PIDs; easoned that if residual CBF defines the hot zones, then we might be able to convert a previously quiet cortical region (i.e., one that did not develop a PID upon stimulation) into a hot zone by artificially bringing the residual CBF in that region to within the critical range, and they did which suggest hot zone location can be dynamic over time; normobaric hyperoxia nearly abolished PIDs (supplied adequate O2 making up for worse neurovascular coupling); hot zones lie within a narrow critical range of hypoperfusion, sufficiently ischemic to render the tissue susceptible, but not ischemic enough to abolish neurotransmission (i.e. electrophysiological penumbra) or to directly
precipitate anoxic depolarization (i.e. core); inducing hypertension may reduce number and size of hot zones
Recent data show that PIDs are indeed triggered by episodic drops in
metabolic supply; focal ischemic penumbra, PIDs predominantly cause hypoperfusion secondary to vasoconstriction in all species studied to date, including mice, rats, and cats, as well as humans. In contrast, when PIDs propagate into the nonischemic tissue, they are associated with a peak hyperemia indistinguishable from SD. The deeper into the ischemic territory, the stronger the hypoperfusion response, and the more severe the loss of perfusion, mildly ischaemic regions have biphasic response; hypoperfusion becomes permanent if depol doesnt recover, and thus core expands; NOS activity may be diminished in ischemic tissue in part due to reduced O2
availability as a substrate for NO synthesis. This is supported by data showing that pharmacological NOS inhibition transforms the CBF response similar to that in ischemic tissue; elevated K has role too
glutamate and infarct volume
glut transporters include glt1 and EAAC1; GLT1 antisense (binds to mRNA and inactivates) but not EAAC1 antisense exacerbated infarct volume; rothstein reported in 2005 that beta lactam antibiotics offer neuroprotection by increasing glt1 expression (also delayed loss of neurons in ALS) and verma 2010 Pre-treatment with ceftriaxone for five days resulted in a significant reduction in neurological deficit as well as cerebral infarct volume after 1h of ischemia followed by 24h of reperfusion injury. It also caused a significant upregulation of GLT-1 mRNA; more glt-1 means enhanced glut uptake; for focal ischaemia can do neuroprotection by targeting NMDAr to target PID and glial glut transporter to inc glut uptake; (nowhere else to put this): campbell 2003, CINC1 is acute phase protein neutrophil chemoattractant, local injury to brain results in IL1b which causes CINC3 expression in brain and liver to produce CINC1, neutrophils mobilised into circulation and recruited to liver, neutrophil recruitment to brain delayed, none in 4 hrs and starting into superficial layers and meninges after 6 (and a bigger dose), anti-CINC1 blocked recruitment of neutrophils to liver and brain, may be via vagal afferents to liver s chemokine communication not found in blood; thus acute brain injury induces rapid innate immune response
focal ischaemia and cell death
increasing evidence implicates apoptosis as important process leading to brain cell death after hypoxicischaemic insults; Inhibitors of macromolecular synthesis and of caspase activity can limit apoptotic death of neurons exposed to hypoxia-ischaemia; In addition, transgenic mice
overexpressing the antiapoptotic gene bcl-2 have
smaller infarcts than their littermates after hypoxiaischaemia; One factor that may promote apoptosis after ischaemic insults is deprivation of
growth factor support. Deprivation may result from damage to neuronal or glial targets responsible for
providing growth factor support. However, tissue concentrations of several growth factors increase in the brain following hypoxic-ischaemic insults, suggesting that there may be either decreased sensitivity of neurons to neurotrophins after ischaemia or increased concentration of neurotrophins are required to counter proapoptotic stimuli, such as free-radical exposure. Addition of exogenous growth factors, such as nerve growth factor or basic fibroblast growth factor, can reduce hypoxic-ischaemic damage. However, while neurotrophins may attenuate ischaemic apoptosis, they may, in contrast, have deleterious effects by enhancing the excitotoxic necrosis induced by ischaemia
raising Ca for necrosis no effect or death, lowering protective, vice versa for apoptosis; blocking protein synthesis, expressing bcl2 or Bax, Bad protects from apoptosis but not necrosis; Oxidative stress may trigger apoptosis following hypoxia-ischaemia. Exposure of neurons to a free radical stress, either by application of H2O2, exposure to UV irradiation or depletion of antioxidant defenses, such as glutathione or superoxide dismutase (SOD), may trigger apoptosis. Free radicals not only may serve as inducers of apoptotic cell death, they may also be a signal in the apoptotic cascade; Persistent impairment of cellular energy metabolism after an ischaemic insult may also play a role in triggering apoptotic neuronal degeneration. Studies in cell culture and animal models of stroke suggest
that inhibition of mitochondrial function by mitochondrial toxins such as 3-nitropropionic acid worsen excitotoxic injury but also can trigger apoptotic neuronal death. Prolonged deficits in mitochondrial function and energy metabolism have been observed after ischaemia-reperfusion and may represent a trigger for apoptotic neurodegeneration after ischaemia. Last, cytokines and inflammatory mediators - IL1b, TNF-a, TGF-b are present in post-ischaemic brain tissue - are also capable of inducing apoptosis; necrosis in core, apoptosis in penumbra: The ultimate choice between apoptosis and necrosis depends on energy levels in the affected cells; Secondary necrosis results from rapid failure to develop the
apoptotic program because of the maintained depletion of apoptosis-requiring energy stores in the core
mitochondria and stroke
A new idea in the “mitochondrial hypothesis” for cerebral ischaemia and stroke is that mitochondria are
transferred from astrocytes to neurons and that this crosstalk may contribute to endogenous
neuroprotective and neurorecovery mechanisms. For example, we know that neurons can release
damaged mitochondria and transfer them to astrocytes. This ability to exchange mitochondria may represent a potential mode of cell-to-cell signalling in the nervous system; hayakawa et al recently showed that astrocytes in mice can release functional mitochondria that enter neurons. Astrocytic release of extracellular mitochondrial particles was mediated by a calcium-dependent mechanism involving CD38 and cyclic ADP ribose signalling. Transient focal cerebral ischaemia in mice inducted entry of astrocytic mitochondria into adjacent neurons, and this entry amplified cell survival signals (e.g., phosphorylated AKT and BCL-XL). Suppression of CD38 signalling by short interfering RNA reduced extracellular mitochondrial transfer and worsened neurological outcomes
When rat cortical neurons were subjected to oxygenglucose deprivation, intracellular ATP levels fell and neuronal viability decreased, as expected; When astrocyte-conditioned media containing
extracellular mitochondrial particles was added to neurons, ATP levels were increased and neuronal viability was recovered; But when extracellular
mitochondria were removed from the astrocyte-conditioned media, neuroprotection was no
longer observed; First, primary mouse cortical astrocyte cultures were labeled with MitoTracker Red CMXRos and extracellular mitochondria particles were collected. Then mice were subjected to focal cerebral ischemia, and 3 days later, extracellular mitochondria particles were directly injected into peri-infarct cortex. After 24 hrs, immunostaining suggested that transplanted astrocytic mitochondria were indeed present in neurons; interfering with CD38 signaling may have suppressed endogenous astrocyte-to-neuron mitochondrial transfer; hese findings suggest that astrocytes may release extracellular
mitochondrial particles via CD38-mediated mechanisms that enter into neurons after stroke; detailed mechanism should be studied further
autophagy in focal ischaemia
another mechanism of cell death/clearance where the cellular process that mediates lysosomal degradation of long-lived cytoplasmic proteins initiated under conditions of differentiation,
nutrient failure, or stress such as oxidative stress, endoplasmic reticulum stress and protein aggregate acculumation. During autophagy, cytoplasmic components are sequestered into double membrane vesicles called autophagosomes, then fuse with lysosomes to produce single membrane autophagosomes, and degraded by lysosomal hydrolases. Recent evidence in a focal ischaemia model with permanent middle cerebral artery occlusion has found that autophagy is activated in the penumbra at various times following ischaemia. Ischaemic postconditioning at the onset of reperfusion
attenuates autophagy, as too does inhibition of the autophagy pathway
histo in core and penumbra
in the early stages of cerebral infarction, neurons of the so-called “necrotic” core display a number of morphological, physiological, and biochemical features of early apoptosis, which include cytoplasmic and nuclear condensations and specific caspase activation cascades. Early activation cascades involve the death receptor pathway linked to caspase-8 and the caspase-1 pathway. They are not associated with alterations of mitochondrial respiration or activation of caspase-9. In contrast, pathways that are activated during the secondary expansion of the lesion in the penumbral area include caspase-9. In agreement with its downstream position in both mitochondria-dependent and -independent pathways, activation of caspase-3 displays a biphasic time course. We suggest that apoptosis is the first commitment to death after acute cerebral ischemia and that the final morphological features observed results from abortion of the process because of severe energy depletion in the core. In contrast, energy-dependent caspase activation cascades are observed in the penumbra in which apoptosis can fully develop because of residual blood supply
delayed neuronal death in global ischaemia
kirino described a form of
neuronal death in the gerbil hippocampus following ischaemia. He called it ‘Delayed Neuronal Death’ because its morphology evolved over a period of 2 to 4 days after brief global cerebral ischaemia. Is this really a delayed form of cell death, or are we really talking about something that has the appearance of delay, but in fact due to secondary processes, rather like the finding of cortical SD in focal ischaemia; In Kirino’s studies and those of others, there was a period early after resuscitation where the morphology
under light-microscopy looked apparently normal.
What is the mechanism underlying this form of death; can use the same pharmacological approach that was used for focal ischaemia: mk801 no effect, NBQX has affect even 24hrs after lesion
recent reports suggest that the amount of N-methyl-d-aspartate receptor and mRNA for the receptor is not very different between the CA1 and CA3 regions - weakens idea of glut excitotoxicty explaining CA1 sensitivity; ischaemia inhibits protein synthesis (one mechanism is in [ca++]i results in eIF2 inactivation, but Ca++ overload transient and protein synthesis shut down for long time so doesnt really explain, prolonged inhib suggest neurons dont have the energy to spare for highly accurate protein synthesis; suggested HSP70 expression altered but immunoreactivity for HSP70 in rat CA1 large but those neurons still die; CA1 mtDNA expression decreases faster than eg CA3, succinate dehydrogenase activity shown to decrease across all CA1 subfield by 7 days, this may result in failure of energy generation in CA1 neurons; An initial increase of intracellular calcium concentration caused by excitatory neurotransmission first damages motor proteins; then the mitochondrial shuttle system is disturbed, followed by a gradual decrease of energy production in mitochondria that eventually causes an energy crisis of the cells, which are increasingly demanding ATP for recovery. These processes finally result in cell death. Thus, this hypothesis cooperates with the previous hypotheses and explains the reason why cell death takes 3 to 4 days after the initial ischemic insult. The delayed neuronal death of hippocampal CA1 neurons seems to be a particular type of necrosis
delayed neuronal death and mitochondria
Mitochondrial calcium overload plays a key role in excitotoxic neuronal injury; overstimulation of neurons evokes the release of proapoptotic proteins from mitochondria; a paradox, however, in that the destruction of mitochondria impairs
the ATP supplies that are essential for activation of energydependent apoptotic pathways. One proposed solution envisions that only a subpopulation of mitochondria undergoes a permeability transition and releases apoptogens, whereas the remaining, undamaged mitochondria respire normally and produce ATP; d cultured hippocampal neurons to investigate the ionic, structural, and functional changes in individual mitochondria that follow injurious NMDA stimulation. The results demonstrate that NMDA overstimulation causes calcium overload only in a subpopulation of mitochondria and that these
mitochondria undergo subsequent swelling, outer membrane rupture, and cytochrome c release. Remaining mitochondria apparently can resume normal functions, thereby creating conditions favorable for the downstream apoptotic reactions that lead to delayed cell death; a few cells died before reperfusion of acute necrosis, but many initially seemed to recover; under EM, After NMDA stimulation, the majority of neurons displayed massive dilation of the endoplasmic reticulum and
swelling of mitochondria, although, importantly, only a subpopulation of mitochondria swells. Many mitochondria changed shape to oval or round, and their diameter increased to 300-500 nm, but not all mitochondria were swollen to the same extent. In
the majority of neurons, there typically were several highly swollen mitochondria scattered among those that were swollen little, if at all. The fraction of severely swollen mitochondria varied
widely from cell to cell but averaged 30-40%; Ca++ fluorescent probe showed A typical time course for [Ca 2+]i during and after NMDA stimulation exhibits an initial fast rise and decay, followed by a sustained elevation that slowly recovers to prestimulus levels after NMDA removal; much elevated mit Ca and phosphorous too; mit buffer Ca++ by forming complexes with it and phosphorous
If the Ca load of an individual mitochondrion within a given neuron does not exceed available Ca 2 buffering capacity, this organelle will be only transiently affected and capable of full recovery. In
contrast, Ca loads that overwhelm buffering capacity will lead to an uncontrolled rise in intramitochondrial free Ca 2, which in turn will trigger an injury response, e.g., a permeability transition and the release of apoptogenic proteins. If enough mitochondria exceed the damage threshold, the cumulative result would be an
effective death signal. In this view, vulnerable neurons would be those that contain a critical fraction of mitochondria, the calcium load of which exceeds the damage threshold. Viewed another
way, the hypothesis predicts that cells with high average [Ca]mito would be expected to be the most vulnerable, because these are more likely to contain the requisite number of damaged organelles
does dnd relate to astrocytes
If we consider the CA3-CA1 connectivity via the Schaffer collaterals, we can bring about CA1
neuroprotection in ischaemia by either toxic KA/AMPA lesion of CA3, surgical lesion of entorrhinal cortex, or blockade of CA3 AMPA receptor activity with NBQX (next 3 studies); Taken together these studies indicate that the integrity of
CA1-CA3 connectivity is vital for the development of CA1 pyramidal cell loss after ischaemia. That is,
rather like focal ischaemia where post-occlusion cortical SD perturbs penumbral tissue, we could propose that after global ischaemia normal neurotransmission via the Shaffer collaterals leads to compromise of vulnerable CA1 cells; other mechanisms proposed: There is recent evidence that differential astrocyte vulnerability in ischaemic injury contributes to CA1 neuronal death. Ouyang et a found that CA1 astrocytes are more sensitive to ischaemia than dentate gyrus (DG) astrocytes. In rats subjected to transient forebrain ischaemia, CA1 astrocytes lose glutamate transport activity and immunoreactivity for GFAP, S100 and glutamate transporter GLT-1 within a few hours of reperfusion, but without astrocyte cell death; porter GLT-1
within a few hours of reperfusion, but without astrocyte cell death (see figures in the paper). Alternatively, oxidative stress may contribute to the observed selective CA1 changes, because CA1 astrocytes show early increase in mitochondrial free radicals and reduced mitochondrial membrane potential. Similar changes were not seen in DG astrocytes. Upregulation of GLT-1 expression in astrocytes protected CA1 neurons; which suggests that greater oxidative stress and loss of GLT-1 function selectively in CA1 astrocytes is central to delayed death of CA1 neurons (link to beta lactam glt1 stuff); Last, the delayed nature of cell death may be related to the time course of altered or abnormal glutamatereceptor mediated calcium influx. For example, switching-off of GluR2 expression in CA1 after the ischaemic insult is translated into the formation of new AMPA receptors lacking the GluR2 subunit. This change in receptor composition increases AMPA receptor-mediated calcium influx in response to endogenous glutamate and enhances pathogenicity
Interruption of the excitatory afferents to the hippocampus by removal of the entorhinal cortex prior to ischemia allows examination of this hypothesis. Groups of adult male Wistar rats were subjected to 20 min of ischemia (four-vessel occlusion) 4 days following a sham procedure, unilateral or bilateral entorhinotomy. CA-1 pyramidal cell survival following ischemia was assessed by light microscopic examination (cell counts) 4 days after ischemia. Compared to control animals unilateral entorhinotomy protected 50% of the CA-1 pyramidal neurons ipsilateral to the lesion, whereas bilateral entorhinotomy resulted in 84% protection; suggested that the protection of CA-1 pyramidal neurons after entorhinotomy is due to interruption of the input to the dentate granule cells, which forms a link in the trisynaptic pathway from the entorhinal cortex to the CA-1
selective dysfunction of CA1 astrocytes contributes to dnd after transient global ischaemia
Patients who survive cardiac arrest face significant neurological disability due to loss of CA1 neurons.
Thus far only hypothermia has been shown to improve neurological outcome in these patients; we investigated the early astrocyte response to ischemic injury comparing the selectively vulnerable CA1 region with the more resistant DG. This study provides evidence for the novel hypothesis that selective hippocampal astrocytic impairment is responsible for the selective loss of CA1 hippocampal neurons following global or forebrain ischemia; evaluated immunoreactivity for GLT1 and GFAP (astrocyte specific filament protein); saw loss of both in CA1 astros post reperfusion, only some GFAP lost in DG and not till later, no GLT1 loss; thee astros not dead tho (took up astro specific dye); CA1 astrocytes were more susceptible to reduction of mitochondrial membrane potential and increased generation of ROS compared to DG astrocytes challenged with the same insult and GLT1 is susceptible to oxidative damage; beta lactam upreg of GLT1 protects against cell death in CA1; our study indicates that early changes in CA1 astrocyte immunoreactivity and function precede the degeneration of CA1 neurons by more than a day so selective vulnerability of CA1 neurons may be secondary to selective ischemia-induced astrocyte dysfunction
AMPA receptors lacking GluR2 are much more Ca++ permeable; In rats, global ischemia leads to reduced expression of GluR2 mRNA in vulnerable pyramidal neurons of the hippocampal CA1 before the delayed cell death; present study was performed in gerbils to test whether the
change in AMPA receptor expression enhances AMPA receptorgated Ca 21 entry into CA1 pyramidal neurons. We first show by in situ hybridization that global ischemia in gerbils, as in rats, leads to a reduction in GluR2 mRNA in these neurons. We then show by intracellular recording and optical imaging of fura-2- injected CA1 neurons that Ca 21 influx through AMPA receptors
is increased after global ischemia. The changes in GluR2 mRNA expression and AMPA receptor function precede neuronal death. These findings indicate that Ca 21 influx through AMPA
receptors lacking the GluR2 subunit may be an important factor contributing to delayed neurodegeneration after global ischemia;ischemia induced neurotrophic factors or stress proteins are plausible candidates for the downregulation of GluR2 expression by reducing mRNA transcription and/or stability. Because some mRNAs and proteins are upregulated in CA1, the decreases in GluR2 mRNA and protein are not simply a result of
a loss of transcriptional or translational capability but seem to result from a regulatory, although maladaptive, change
so why is CA1 so vulnerable?
NBQX delayed effect? Acting on 2o mechanism
of post-ischaemic CA3àCA1 neurotransmission
* GLT-1 effect? We appear to have selective
vulnerability of CA1 astrocytes
* GLU-R2 hypothesis? Reduced post-ischaemia
expression of mRNA for GLU-R2 AMPA subunit
* “Weak excitotoxicity”? An interaction between
“energy failure” (mitochondria link) and altered sensitivity at NMDAr; both focal and global involve changes in glial glut uptake, but focal is NMDAr dependent and global is non NMDAr mediated; GNN and DND are really more a phenomenon of secondary delayed insults than a true delayed process of cell death; If it takes 30 - 60 minutes of MCA occlusion to produce a stroke, then why does it take only 5 minutes of no cardiac output to injure the hippocampus?; Is this problem really about anatomical differences, and differences in
the repertoire of induced secondary mechanisms? (hint: the latter)
Zn and CA1 vulnerability
Excitotoxic mechanisms contribute to the degeneration of hippocampal pyramidal neurons after recurrent seizures and brain ischemia.
However, susceptibility differs, with CA1 neurons degenerating preferentially after global ischemia and CA3 neurons after limbic seizures. Whereas most studies address contributions of excitotoxic Ca 2 entry, it is apparent that Zn2 also contributes, reflecting accumulation in neurons either after synaptic release and entry through postsynaptic channels or upon mobilization from intracellular
Zn2-binding proteins such as metallothionein-III (MT-III). Using mouse hippocampal slices to study acute oxygen glucose deprivation (OGD)-triggered neurodegeneration, we found evidence for early contributions of excitotoxic Ca 2 and Zn2 accumulation in both CA1 and CA3, asindicated bythe ability of Zn2 chelators or Ca 2 entry blockersto delay pyramidal neuronal deathin both regions.However, using knock-out animals (of MT-III and vesicular Zn2 transporter, ZnT3) and channel blockers revealed substantial differences in relevant Zn2 sources, with critical contributions of presynaptic release and its permeation through Ca 2- (and Zn2)-permeable AMPA channels in CA3 and Zn2 mobilization from MT-III predominating in CA1. To assess the consequences of the intracellular Zn2 accumulation, we used OGD exposures slightly shorter than those causing acute neuronal death; under these conditions, cytosolic Zn2 rises persistedfor 10 -30 min after OGD,followed by recovery over40 - 60 min. Furthermore,the recovery appearedto be accompanied by mitochondrial Zn2 accumulation (via the mitochondrial Ca 2 uniporter MCU) in CA1 but not in CA3 neurons and was markedly
diminished in MT-III knock-outs, suggesting that it depended upon Zn2 mobilization from this protein; blocking the MCU attenuates mitochondrial swelling; neurons show mitochondrial swelling
with release of cytochrome C into the cytosol beginning within hours of ischemia, before caspase-3 activation and with the appearance of TUNEL-positive cells and neurodegeneration with
prominent DNA fragmentation occurring over several days
how astrocytes protect CA3 neurons
In the hippocampus, delayed neuronal death is normally seen in neurons of the CA1 region but not in those of the CA3 region. Astrocytes have been reported to play multiple supporting or pathological roles in neuronal functioning. While evidence indicates that astrocytes could exert neuroprotective effects following ischemia, the
possible underlying mechanisms remain unclear. We aimed to investigate the roles of astrocytes in the process of delayed neuronal death following transient forebrain ischemia. L-α-aminoadipic acid (L-α-AAA), an astrocyte-selective gliotoxin, was injected into the hippocampal CA3 region of rats through a cranial window to selectively damage
astrocytes. Immunofluorescence staining of glial fibrillary acidic protein (GFAP) was used to evaluate the effect of L-α-AAA on astrocyte numbers. Three days after the L-α-AAA injection, transient forebrain ischemia was induced by a modification of the four-vessel occlusion procedure. Seven days after transient forebrain ischemia, hematoxylin-eosin staining was performed to reveal the morphology of hippocampal pyramidal neurons. In rats with ischemia and reperfusion, regional cerebral blood flow (rCBF) and change in intracellular Ca2+ concentration ([Ca2+]i) were separately measured in CA1 and CA3 regions. L-α-AAA injection significantly decreased the number of astrocytes in CA3, but did not affect the pattern of rCBF changes upon ischemia/reperfusion. Seven days
after transient forebrain ischemia, in rats receiving L-α-AAA, delayed neuronal death comparable with that in CA1 was observed in the CA3 region. In addition, the pattern of increase in [Ca2+]i due to transient forebrain ischemia was completely changed in the hippocampal CA3. The loss of astrocytes induced a persistent increase in [Ca2+]i
in the CA3 region following transient ischemia, similar to what is observed in the CA1 region. Our study indicates that astrocytes in the hippocampal CA3 region exert neuroprotective effects following transient forebrain ischemia and act by suppressing the intracellular Ca2+ overload; they imaged the calcium deep inside the brain; Consistent with our results, Ouyang et al. (2007) reported that selective dysfunction of hippocampal CA1 astrocytes contributes to delayed neuronal damage after transient forebrain
ischemia. However, these authors focused on the loss of glutamate transporter-1 function in CA1 astrocytes; Astrocytes can upregulate pentraxin 3 to maintain blood-brain-barrier integrity by regulating vascular endothelial growth factor-related mechanisms (Shindo et al., 2016).
Astrocytes can also mobilize functional mitochondria and transport them to neurons after stroke to contribute to the neuroprotection and functional recovery of neurons (Hayakawa
et al., 2016); Another important finding of our study was that the intracellular Ca2+ responses in CA1 and CA3 regions after transient forebrain ischemia were significantly different. While the [Ca2+]i
in hippocampal CA1 persistently trended upward after transient forebrain ischemia/reperfusion,
this effect was not seen in the hippocampal CA3. Furthermore, delayed neuronal death occurs in the hippocampal CA1 region after transient forebrain ischemia, but not in the CA3 region (Kirino et al., 1984). This contrast in patterns of [Ca2+]i
increase following ischemia may be one of the reasons for the selective vulnerability of hippocampal CA1 neurons to ischemia. The differences between the hippocampal CA1 and
CA3/dentate gyrus (DG) regions have been extensively studied; For instance, the expression of glutamate transporter-1 and heat shock protein 70 in the CA1 region are higher than in the CA3/DG region; mechanism for astrocyte Ca++ reduction and hippocampal CA3 astrocytes may have a special, enhanced ability to regulate glutamate and Ca2+ toxicitymight be removing glut from ecf
galectin-3 in dnd
The ischemic damage in the hippocampal CA1 sector following transient ischemia, delayed
neuronal death, is a typical apoptosis, but the mechanism underlying the delayed neuronal
death is still far from fully understood. Galectin-3 is a β-galactosidase-binding lectin which is
important in cell proliferation and apoptotic regulation. Galectin-3 is expressed by microglial
cells in experimental models of adult stroke. It has been reported that activated microglial
cells are widely observed in the brain, including in the hippocampal CA1 region after transient
ischemic insult. In the present study, time course expression of galectin-3 following transient
forebrain ischemia in gerbils was examined by immunohistochemistry, combined with Iba-1
immunostaining (a specific microglial cell marker); Following transient ischemia, we observed a transient increase of galectin-3 expression in CA1 region, which was maximal 96 h after reperfusion. Galectin-3 expression was predominately localized within CA1 region and observed only in cells which expressed Iba-1. The galectin-3-positive microglial cells emerge after the onset of neuronal cell damage. Expressions of galectin-3 and Iba-1 were strongly reduced by hypothermia during ischemic insult. Prevention of galectin-3 and Iba-1 expression in microglia by hypothermia has led us to propose that hypothermia either inhibits microglial activation or prevents delayed neuronal death itself; galectin-3 is pro-apoptotic
