Wound Healing and Inflammation + MISC Stuff Flashcards
Difference between tissue-resident and monocyte derived macrophages?
Tissue -> early in embryogenesis, erly sorce of proinflammatory cytokines
Monocyte -> differentiate in tissue and reside in ECM (main cell), monocytes promote vascular regrowth
Difference between M1 and M2 cells?
M1:
-activated by infectious agents or proinflmmatory cytokines (IFN-gamma/TNF-alpha)
-debride via phagocytosis
-produces proinflammatory cytokines and PG
M2:
- activated by antiinflammatory cytokines (IL-4, IL-13, IL-10)
- aids n wound repair -> secretes GFs + fibroblasts to produce collagen
Both M1 and M2 secrete collagenase/elastase)
What does it mean to have loci considered “in linkage?”
If genetic distance between marker allele and mutation small enough that mutation is transmitted with trait between generations
What type of wound is best w/bioscaffold & synthetic matrix?
Chronic indolent wound
-acts as comp. substrates for MMP, collagenase
-allows proliferation of ECM
What are some main changes in metabolism of burns?
- Initially hypometabolism “ebb phase”
-basal energy, expenditure during flow phase increases >100% compared to pre-burn - post-burn adipose-> 1/ of energy requirement, protein catabolism 30%
-“Burn diabetes” -> glucose uptake increased from gluconeogenesis and insulin resistance
anerobic glycolysis very high in endothelium and fibroblasts!!
What % of mesencyhmal cells given systemical injection sucessfully home and migrate to site of injury?
What product should be added to it to improve local effect?
<5%
PRP of fibrin gel
For radio, CO2 laser, monopolar cautery char penetrates skin peripheral to skin edge ____mm?
Radio: 0.17mm
CO2: 0.215
Monopolar: 0.255
(For PRP) When does platelet release alpha graules stores & how much is released within 1 hour?
within 10 min
95% is eleased within an hour so give the PRP quickly!
What does each of the following code for?
mRNA
rRNA
tRNA
Small nuclear RNA
Small nucleolar RNA
mRNA - protein sequences
rRNA-mitochondrial ribosomal subunits for translation
tRNA - AA binding sites
Small nuclear RNA - units of spliceosomes
Small nucleolar RNA -molecules in RNA modification
What are advantages and disadvantages of retroviral useage?
Adv: nonimmunogenic, integrate with host genome for long-term expression
Disadv: target cell must be dividing before retrovirus integrates, site of integration random, can cause deleterious effects
Define:
point mutation
missense mutation
nonsense mutation
synonymous mutation
frame shift mutation
microsatellites
point mutation: chnge sequence or length, non-synonymous
missense mutation: change to AA codon -> alter protein structure
nonsense mutation: replace AA codon with stop codon/ends protein sequence
synonymous mutation: changes genetic sequence but still codes for right AA/protein not changed
frame shift mutation: Add or deletes single/multiple base pairs so all are shifted by #
microsatellites: -6 base pair motifs repeated # of times
Know equations of RER (2)
Know protein maintenence loads dog/cats
RER=30BW(kg) +70 or 70(BW(kg))^0.75
How is autologous conditioned sera (IRAP) made?
- Whole blood -> inject into chanber with borosilicate beads
- Coagulates, incubate it at 98.6F for 6-24 hour
- Leukocytes adhere to the beads and it induces signalling & expression of IL-1Ra
- Centrifuge it to isolate the serum and then give it IA
What is difference in collagen prevalence with unwounded vs wounded dermis?
Unwounded: 80% Type 1 / 20% type III
Wounded: Mainly III
What 2 main factors are present in collagen synthesis & what are their functions?
TGF-B
- synthesis T1 collagen
- decrease production of MMPs
-increase production MMP inhibitors
IL-10 (weeks later)
-limits fibroblast recruitment
-decreases production of interferons and PF4
When does granulation tissue appear?
~7 days
With moist wound healing, what dressings would be appropriate for:
Dry
Mild to moderate
heavily exudative
Dry:
water based hydrogel
Mild to moderate:
hydrocolloid sheet, polyurethane foam, maltodexterin polymer flakes (all form gel layer)
heavily exudative:
Ca alginate
If infected hypertonic gauze
Describe major changes in pulmonary system with burns
CO-> decreased O2 carrying capacity, decreased delivery
HCN-> prevents cell respiration
HCl -> irritating to resp. mm, laryngospasm, bronchospasm
Increased pulm vascular permeability and vasoconstriction
atelectasis, deactivation of surfactant
cytokines etc-> apoptosis too from GI endotoxin
What height do dog ears typically regress?
What are techniques to resolve?
<8mm
apex cutaneous suture
horizontal oblique
excise triangles, fusiform, arrowhead, “fudging”
central removal
end removal
rule of halves
Translation goes in which direction?
What are the stop codons?
5’->3’
UAA, UAG, UGA
- When does fibroblast migration occur?
- What factor synthesizes definitive ECM?
- What type of procollagen is formed?
- What cytokines stimulate fibroblasts to migrate?
- 3-5 days post wounding
- Fibroblast growth factor
- Type I and III
- PDGF, TGF-B, and epidermal GF from platelets and macrophages
NB: FGF has many functions but it increases production of fibroblasts, not necessarily their migration
What are the main functions of nitric oxide?
-regulate vascular tone
-produce vasodilation and relaxation
-antagoize vasoconstriction effect of angiotensin II, ROS
-decrease platelet aggregation and leukocyte adesion
-anti-inflammatory effects on GI
-increase production of pro-inflamm cytokines
-induces TH-1 at low concntrations and inhibits them at high concentrations
Which IL-1 is proinflammatory?
Which is Anti?
IL-1Ra -> anti
IL-1B -> Pro
How do chemokines work?
Example?
How are they categorized?
Attraction of cells accross concentration gradient during embryonic development, wound healing, angiogenesis, and inflammatory responses
They are categorized by cyteine residues (e.g. IL-8 = CXCL8)
What are the primary functions of macrophages in wound healing?
- Cytokines IL-1/6/8, TNF-alpha, FGF, EGF, TGF-beta, PDGF
- induce neutrophil apaotosis at 48-96hr
-release metalloprotinases MMP 1/2/3/9 whih degrade ECM to help cells move accross wound
Neutrophil migration and adherence is primarily promoted by which cytokines/mediators?
What other factor amplifies chemotaxis and (+) macrophage, keratinocyte and fibroblast expression?
- PDGF, IL-1, TGF-beta, c3a/c5a, LTB4
-TNF-alpha
Where does platelet-activating factor come from?
Functions?
From phospholipase A2
-Stimulates arachidonic acid release and increases eicosanoids
-increases affinity of surface integrins to enhance adhesion
- platelet aggregation and degranulation
- increased vascular permeability
-bronchoconstriction
What are the main effects of ROS?
-Antibacterial defense, wound debridement, cell signalling, pathologic tissue (??word)
- Homeostasis - generates ATP via respiratory burst / burst helpful to defend from bacterial invaders
- pathologic -> from frustrated phagocytosis that releases damaging agents into extracellular space -> produces ischemia-> ROS
What are examples of positive acute phase proteins?
Negative?
Positive: serum amyloid P, complement protein, coag factors, CRP, Serum amyloid A, kallekrein
Negative: albumin, transferrin, apolipoprotein a
Eicosanoids - what is the precursor?
Made from what via which enzyme?
Cell membrane phospholipids-> via phospholipase A -> arachidonic acid (precursor)
AA-> prostaglandins (via COX) and leukotrienes (via LOX)
Bladder gets 100% of strength within ___ days?
Collagen synthesis normal after ___ days?
Re-epithelializes in ___ days?
21 days
70 days
2-4 days
What are all of the factors released by or stimulated to be released by platelets?
PDGF
TGF-beta
IGF-1
EGF
fibronectin
fibrinogen
thrombospondin
vWF
Factor 8
Name the functions of neutrophils and the cytokines produced
phagocytosis
release superoxide radicals
form neutrophil traps
secrete MMPs
produce cytokines (IL-1/1a/1b/6, TNF-alpha)
What is the most significant PRR (pattern recognition receptor) and what cytokine does it influence?
Toll-like receptors (TLR)
NFkappa-beta causes the leukocyte to release cytokines
Negative pressure wound therapy:
1. What are the size of pores in foam?
2. Standard pressure?
3. What pressure for gauze?
4. Pressure for less-dense tissues?
5. Pressure over grafts?
400-600um pores
-125mmHg standard
-80 for gauze
-75-100 for less dense tissue
-65-75 for grafts
Golden period is how long it takes bacteria to reach ___#s?
10^5 per gram of tissue or mL of exudate
Give the main examples of:
vasoactive amines
pro-inflammatory cytokines
anti-inflammatory cytokines
Vasoactive- histamine, serotonin
pro-inflam-> TNF, IL-1, IL-6, Chemokines (IL-8)
Anti-inflam -> IL-10
Where are kininogens formed?
What are kinins?
What is seen on coags if kinin deficiency?
Formed in liver then circulate
they are vasodilatory proteins
prolonged APTT w/normal coag factors
Partial pressure of O2 must be above what to kill bacteria effectively?
> 40mmHg
List the tissues in order of High-> low for residual wound strength after healing:
colon, bladder, stomach, skin
Bladder>Stomach>colon>skin
What is the function of telomerase?
What is it implicated in?
reverse transcriptase - it adds dna sequences to repeat to 3’ end of DNA in telomere region at end of desmosomes
It is implicated in oncogenesis and in aging
What is the difference between a PAMP and a DAMP?
PAMP = pathogen-associated (exogenous foreign stuff)
DAMP = danger-associated ->endogenous stuff like fibrinogen/cell damage
Both bind to TLR and increase inflammation
Collagenase breaks collagen in how many days after wounding?
1-2 days
On distal limbs, loose skin is more available in which direction?
Circumferential
What activates the classical, lectin, and alternative complement pathways?
All pathways converge at cleavage of which component?
classical-> immune complexes (antibodies)
lectin-> hepatic lectin proteins bind carbs
alternative-> foreign microbes
All merge at C3
What are the 3 collagen types in GI submucosa and %s?
I (68%)
III (20%)
V (12%)
What are the main differences in function between prostacyclin (PGI2) and thromboxane (TXA2)?
PGI2 causes vasodilation and inhibits aggregation of platelets
TXA2 causes vasoconstriction and promotes aggregation
RNA silencing ends with what two versions of RNA and what do each do?
siRNA -> cleave complimentary RNA
miRNA -> interfere with translation
Burn shock comes from what moderators?
chemical pain, mediators like PGs and kinins
sensitize local nociceptors into a hyperalgesic state
What are main effects on CVS from burns?
- hypovolemia, vascular dysfunction, generalized edema (histamine), hyperviscosity
- decreased left ventricular contractility because of Ca leakage to cytoplasm and Na accumulation in cytosol of heart muocytes
What are tachykinins?
Example/function?
neuropeptides released from peripheral neurons after stimulation or trauma to sensory neurons
e.g substance P
-promotes transmission of pain/inflammation
-vasodilation and edema, leukocyte chemotaxis +adhesion
What unsaturated fatty acid is essential for dog diets?
What is carnitine?
Linoleic acid
Carnitine is a quaternary ammonium compound biosynthesized from lysine and methionine. It is a cofactor for transport of long-chain FA accross mitochondrial membrane to liberate energy
Can improve function + energy metabolism
What is the benefit of silver dressings?
Warning?
Or appearance?
Broad spectrum antibiotic action
- bound elastase decreases concentration of TNF-alpha, IL-8 and inhibits free radical formation
WARNING: ONLY USE IN INFECTED WOUNDS
Silver exudate when change bandages -> seen with pseudomonas so lavage thoroughly
Describe the main changes in uncomplicated starvation:
Insulin decreases, glucagon increases, fatty acids increase-> become ketones, glycogen decreases
gluconeogenesis increases
glycogenolysis increases
Initially catecholamines decrease, but when sick sympathetic system increases them
Results -> insulin resistance, decreased cardiac output, increased vascular resistance, increased protein catabolism, malnutrition
Most calories in dry food ___ kcal/g
carbs/protein -> ____kcal/g (dry matter)?
8.5 kcal/g
3.5 kcal/g
What are the main effects of histamine?
H1 -> Vasodilation, increased vascular permeability, nonvascular smooth muscle contraction
H2 -> smooth mm contraction, neurotransmission, gastric acid production
Wounded skin is what % of normal at:
14 day
3-4 wk
several months
1 year
5-10%
25%
70-80%
80%
What is the difference between cat and dog wound healing?
Cats have lower cutaneous perfusion first 1 week and lower wound breaking strength 1st week
-cats heal more by contraction vs dog is central pull/epithelialization
When on average does granulation tissue appear in dogs? Cats?
What surgical technique can (they say) affect wound healing in cats?
Dog 4.5 days / 6.3 days cats
removal of SQ tissue -> decreases granulation tissue production and wound contracture in cats
Describe main events that occur within maturation phase of wound healing?
- cross linking andreorientation of collagen bundles
-maturation of epidermal layers
-HA increases, fibronectin decreases, collagen increases
-collagen type I increases as type III decreases Type III only 10% in scar) - As tension occurs, collagen bundles align with the stress, crosslink and increase diameter
- fibroblasts stimulateed to differentiate into myofibroblasts in stiff matrix
Why do dextrose and amino acids for parenteral nutrition need to go into cranial vena cava?
Does lipid emulsion have to go in the cava?
- it’s hyperosmotic/causes peripheral phlebitis
- no, lipid is isoosmotic
What are mechanisms Negative wound therapy works? (to help wound healing?)
-improve perfusion
-reduce edema
-remove exudate
-increase tissue pressure
-stimulate granulation tissue
-decrease bacterial colonization
-decrease the pO2 within the wound
Mesenchymal cells defined as having what qualities?
-adhere to the tissue culture plastic and are spindle-shaped
-form colonies of cells from single parent cell when cultures
- express specific markers that are excluded from hematopoeitic lineages
- differentiate into osteoblasts, adipocytes and chondrocytes
What is chitosan made from?
What does it do?
Crab shell
Enhances the function of inflammatory cells and increases production of granulation tissue through upregulation of TGF-B, PDGF and fibroblasts
What type of dressing is honey?
How is it effective?
Hyperosmotic agent
-produces H2O2 from glucose oxidase
-hyperosmotic effects
-lowers pH
+/- B and T cell stimulus, phagocytic stimulus
+/- cytokine release from monocytes
Enhances granulation tissue and decreases edema/inflammation
Explain 3 zones of injury with burns
Zone of coagulation (no viable tissue)
Zone of stasis - decreased deformability of RBC, decreased vascular lumen, decreased perfusion, increased vasc permeability
Zone of hyperemia - primary area of inflammation, vasodilation via NO
What temp does failure of the cell membrane occur at?
Epidermal necrosis at what temp?
40-44C
60C for 1 sec = necrosis
Breifly describe the steps of leukocyte extravasation
- leukocyte ligands interact with endothelial selectins
- Integrins on leukocyte surface are upregulatedby proinflammatry mediators and adhesion molecules
- Adhesion molecules near the interendothelial cell junction interact with integrin and facilitate migration
- PECAM-1 facilitates transendothelial migration and transmigration through basement membrane
once bound to ECM the leukocytes secrete enzymes directing movement to chemoattractants
Where is carbon monoxide formed?
Where is hydrogen sulfide formed?
Are they pro or anti-inflammatory?
CO is from when heme is converted to bilirubin
HS is made during cysteine metabolism or as a byproduct of GI flora
Overall both are anti-inflammatory
Of the CD4+ cells:
What stimulates TH1/TH2
What are their functions?
TH-1 is from IFN-gamma and IL-12
-maximizes macrophage function
-stimulates proliferation of CD8 cells
-Produces IFN-gamma and IL-2
TH2 is helminthic infections/allergies
- Produces IL-4, IL-5, IL-10, IL-13 -> these improve M2 function and increase IgG1 and IgE, eosinophils
-Produce IL-4/10 that inhibits TH1 proliferation
- Epithelialization happens primarily from where?
- Describe the process?
- What stops migration?
- EGF, TGF-alpha, FGF-2
- Keratinocyte integrins interact with ECM fibronectin
-Keratinocytes loosen from basal lamina and mobilize
- Keratinocytes and endothelial cells secrete MMP to break down ECM and phagocytose
-cells behind phagocytic cells proliferate and advance - Contact inhibition by other epithelial cells
For angiogenesis:
- what is the main factor at play?
- What blood parameter does the endothelial response rely on?
- vascular network forms in what timeframe?
- VEGF
-reliant on paO2 - within 4-6 days
What are the 5 main steps of the proliferative phase of wound healing?
angiogenesis
fibroblast migration
collagen synthesis
contraction
epithelialization
Explain the difference between autocrine, paracrine, and juxtacrine signalling
auto - by specific cells acts on that cell or cell of same type
para - by one cell type acting on different but local cell types with the right receptors
juxta - contact-dependent signalling by cells in contact with others or between molecules and ECM glycoprotein
How do you achieve <5psi vs 5-8PSI for irrigation of wounds?
<5 high volume single-lumen port spiked into fluid bag
5-8 -> 18g needle and 35mL syringe (some say 16-22g and 1L bag at 300mmHg pressure)
What is the platelet concentration difference between PRP and peripheral blood?
Explain the difference between centrifugation and the gravity-filtration method?
PRP is 3-5 fold higher
centrifugation -> anticoagulated whole blood
- centrifuge at low force
-second spin gives a buffy coat with large # platelets
Gravity: 60mL whole blood + anticoagulant in a reservoir bag with a capture solution, to increase the size of platelets, then pass through a filter and flush with saline.
Explain the different degrees of burns
1st - only epidermis +erythema (no blister)
2nd - full thickness into dermis -> fluid extravasation = blister
3rd - through dermis to underlying SQ
4th - down to fascia or muscle
5th -> bone
What are the different wound closure methods?
Primary / first intention - sutured wound
delayed primary -> 3-5 days after wound but before granulation tissue
secondary - closed after granulation tissue
second intention - without suture / allow contracture and epithelialization
Neutrophils are in wound primarily during what time frame of healing?
When do macrophages arrive?
Neuts -> 24-48hr
Macs 48-72hr
Difference between totipotent and pluripotent?
specific features of adult stem cells?
Advantage of using induced pluripotent ?
Totipotent -> can become cells from each germ layer and extraembryonic tissues
Pluripotent - each germ layer but not extraembryonic
Adv of induced pluripotent: easily acquired from living donors/unlimited cells for autologous use
Disadv: retroviral/lentiviral vectors to induce can cause mutagenesis or oncogenic transformation
What are the major GI changes from burns?
(not burns of the GIT, but of severe burn patients)
can cause SIRS or MODS
translocation of gut bacteria
Decreased GI motility dur to NO
increased apoptosis of GIT cells
oxidative stress on the liver -> increased catecholamines -> hepatic lipidosis
Describe the main changes that occur in hemostasis phase of inflammatory phase of wound healing
Keratinocytes release IL-1
initial vasoconstriction
platelets bind to IV/V collagen endothelium -> coag cascade and fibrin -> platelet factors, TXA2, vasoconstriction, attract leukocytes
Platelets and most cells produce histamine and serotonin to drive vasodilation and increase permeability for other cells
Is PO2 or saturation of Hb more important for wound perfusion?
- pO2 -> to diffuse accross large intercappilary distances
Give examples of proresolution eicosanoid functions?
Lipoxins
-halt neutrophil infiltration
-activate macrophage phagocytes
-increase clearance of phagocytes
-block secretion of TNF alpha
-resolvins and protectins (Omega-3) decrease inflammation
What produces IL-10?
Functions?
CD4, TH2, monocytes, B cells
inhibits translocation of NFk-B which decreases production of cytokines
downregulates TH1 cytokine production
Limits inflammatory response to normal GIT bacteria
increases TNF-a receptor circulation
inhibits antigen presentation by macrophages and dendritic cells
What is the other name of TNF, where is it mainly produced?
Functions?
cachectin -> from M1
produce cytokines, ROS, chemotaxins, endothelial adhesion molecules
activate NK cells, proliferation of CD8, T-cell apoptosis
What are effects of leukotrienes?
-chemotactic, neutrophil activator
-vasoactive (more than histamine)
-neut extravasation and degranulation
- increased production of free radicals
Peptidoleukotrienes -> provoke vasoconstriction, bronchoconstirction and increase venule permeability
What are % disribution for important (compartments?) for water?
64% intracellular
22% interstitum
7% plasma
7% transcellular
How does bacteria impair wound healing?
Endotoxins and proteases make the granulation tissue friable, decrease fibroblast proliferation, disorganize the collagen
break down the collagen
inhibit keratinocyte migrations
What is the recommended concentration for chlorhexidine?
povidone-iodine?
Chlorhex = 0.03-0.1% 1:40 dilution of stock 2%
Iodine = 0.1-0.01% (1:100-1:1000 dilution of stock 10%)
What is the main difference between GI and skin healing in proliferative phase?
Fibroblasts and smooth muscle cells synthesize collagen/elastin w/smooth m. cells more absolute collagen than fibroblasts
What are the % strength of anastomoses in first 48h of:
esophagus
stomach
colon
SI
Esoph (37%); gastroduodenal (64%); SI (70%); Colon (72%)
What is the main function of IL-6 and what is its use in clinical evaluation?
IL-6 initiates hepatic synthesis of acute phase proteins and proliferation of lymphocytes. Can be used to predict post-op infection, sepsis-mortality and recurrence of adhesions
Vascular perfusion <___paO2 impairs angiogenesis?
<10mmHg
Fascial healing is what type of collagen?
Type 1
