---start path.lec.04.11.97---

pathology
the inimitable dr weber


note: i am having a Bad Day.

dr weber wants to know if we are all going to the square dance tonight.
he's going to be the caller, he says. do-si-do from 8 to midnight.

the exam is almost done. 40% is dr dambach, 40% is dr weber, 20% is 
chacko, which will be essay. dambach and weber is multiple choice. dr 
weber says exam is a gift.

so far we've left one aspect of acute inflammation up to now...
the exam stops with granulomatous inflammation. today and monday stuff 
will be on the final. there is a fair amt of material showing basic 
understanding of the whole process though.

for monday, he has a little homework for us. in order to continue 
discussions with healing, he's made up like 6 problems, and divided us up 
into different groups, and we'll go through them on monday. then he'll go 
into detail about healing processes on different organs.

ok. acute inflammatory response when it occurs, w/in first 24-48 hrs 
starts very active process that ends up with regeneration and repair of 
injured tissue. healing involves regeneration and repair. regeneration: 
tissues will replace the damaged tissue, can only occur in those tissues 
where cells are able to divide. repair implies that the damaged area is 
replaced by fibrous CT. the severit of the response and amt of 
destruction that occurs determines how much repair takes place and how 
much collagen is deposited, how muhc of a scar. the worse the damage, the 
more likely you get a big scar.

before we describe how a wound heals, we need to get into our heads 
something else...how do you classify tissue components? cells are either:


labile
stable
or
permanent

with respect to ability to divide.
labile cells in normal tissue are always turning over, always dividing. 
in surface epithelium, mucosal linings, when you have a surface 
inflammatory rxn, these cells are quite capable of regenerating. bone 
marrow cells, too.

stable cells: parenchymal. liver, kidney, adrenal, etc. in these organs, 
few cells are in the cell cycle. little routine cell division. but they 
are capable of entering the cell cycle. comparing liver, pancreas, 
kidney: which is most capable of entering cell cycle? liver. in kidney, 
tubules can to some extent. pancreatic acinar cells have slight 
regenerative capacity. but islet cells, once eliminated, are permanently 
lost. so stable cells usually don't divide, but can be triggered to do so 
by inflamm process

permanent cells: don't divide. brain neurons and myocytes of cardiac 
muscle. skeletal muscle is kind of permanent, has very very limited 
regeneration ability. when these tissues are damaged, they repair. 

smooth muscle has limited regenerative capacity...more considered stable 
than permanent.

healing process has two categories:

first intention healing: primary union 

second intention healing: secondary union

the difference is, first intention healing produces very little 
recognizable scar. second intention involves a lot of tissue destruction, 
a lot of repair, and considerable scar formation.

main effect is on amt of scar tissue formed.


for discussion purposes, we will make big surface wound in skin of an 
animal. massive wound. divide the wound into a few sections to get an 
idea of time frame.

a few years ago, someone told dr weber a wound heals in pie shaped 
sections - that is not true. this diagram is for discussion only.

0-48 hrs: whole area will fill with blood clot and fibrin. we will get a 
neutrophilic infiltrate at the border of the wound (occurs in viable 
tissue only. can't get inflammatory response in a blood clot). we will 
see proteinaceous edema fluid coming in with the neutrophils, and also an 
influx of mononuclear cells eg monocytes - but neutrophils will 
predominate. dilation of blood vessels, etc. all acute infl rxn stuff we 
discussed will occur at edges of wound.

48-96 hrs: at this point, the number of neutrophils is drasticly 
decreased. we're assuming wound isn't contaminated with bacteria/debris. 
so, no infxn or foreign body. so neutrophils drop off, and now we see 
mostly macrophages. healing can't take place w/o the blood/fibrin clot 
being removed. the mphages phagocytose the dead/dying neutrophils and the 
clot, to prepare wound bed for healing, so that initial extracellular 
matrix can form there. the initial extracellular matrix - extracellular 
matrix has three categories: proteoglycans, adhesive glycoproteins, and 
structural fibrous component. the proteoglycans is a gel matrix which 
contains things like heparan sulfate, dermatan sulfate, hyaluronic acid, 
etc. this forms in the area where mphages have cleared out the clot. 
these proteoglycans and glycosaminoglycans are important b/c they allow 
the immigration of the fibroblasts and endothelial cells. this gel like 
matrix doens't have a lot of structure. in this area we also get the 
adhesive glycoproteins eg fibronectin, laminin, thrombospondin. where do 
they come from and why? well fibronectin in a wound originally comes from 
the plasma, eg the edema carries it. it is important for coating 
everything in the vicinity of the wound healing. coats fibrin, collagen, 
elastin, cells, and it allows motility and directional movement of cells 
with fibronectin receptors. stimulates further differentiation of cells. 
is crucial for cell repair process. laminin does similar thing and 
eventually is one of prime constituents in basement membranes of new 
blood vessels. acts like fibronectin, though. thrombospondin comes from 
platelets. so there are a lot of platelets in the blood clot, and 
thrombospondin is released, and allows cell population to get directional 
movement, motility and further differentiation. 

the structural fibrous components are your fibrillar collagens. these 
really come in later. but, anyway, for wound healing in normal tissue we 
see type I, III, and V usually. these are fibrillar structures. in an 
early wound, mostly type III, then replaced by type I. type V is the kind 
that allows interstitial attachment to other structures in the tissue you 
are in. type IV is found mostly in basement membranes. but these 
collagens come in later.

so we have the gel matrix formed, and the adhesive glycoproteins. we need 
that for the next step to occur, which is:

96-140 hrs: immigration of fibroblasts and endothelial cells into the 
matrix.
we start to have the ingrowth of newly forming blood vessels, with lots 
of anastomoses.realize wound edges have lots of damaged blood vessels. 
when they start to grow, we have some basement membrane which needs to 
get broken down by various collagenases, but then the viable endothelial 
cells start dividing, and push buds of endothelium into the ECM which has 
formed in the wound bed. so most of the mitosis occur behind this leading 
edge of newly formed cells. grow about .1 to .3 mm/day, these vessels. 
this is a pretty fast rate for a little blood vessel even though it may 
not seem fast to you. so the fibroblasts start going in, also, and they 
start laying down those structural fibrous things we mentioned...collagen 
type III first.  the appearance of the wound bed is very characteristic, 
and is called "granulation tissue". if you removed the clot, and looked 
at wound surface, it appears granular. it's moist, red, and if you rubbed 
over it with gauze, it bleeds very easily. now, remember, we discussed 
granulomas the other day. you wanna make sure that you understand the 
difference. granulation tissue is totally different. think of granulation 
tissue as fibrovascular tissue, if that helps. it is newly forming blood 
vessels and lots of fibroblasts and newly deposited ECM. so, vessels are 
growing in, anastomosing, more matrix is deposited...healing only takes 
place in viable tissue, remember. gradually this granulation tissue fills 
in the defect, replacing the clot/fibrin. at the surface of the wound bed 
we always have  alot of macrophages and fluid, etc. the mature part of 
the wound is deeper, and it moves on up to fill in the defect. now, once 
you start granulation tissue forming, the fibroblasts start pumping out a 
lot of fibronectin. so early in wound healing, the fibronectin came from 
plasma, but later, it is formed by fibroblasts and also to some extent 
from smooth muscle and endothelial cells. where do these fibroblasts come 
from? in a tissue like this, most probably come from "undifferentiated 
mesenchymal cells" which are scattered throughout the body's CT and which 
can differentiate along multiple pathways depending on environment. so. 
granulation tissue matures during this time.

140 hrs+ - in an uncomplicated wound healing, as wound healing matures, 
what are the changes compared to the 96-140 hr period? before, there are 
still lots of macrophages, still edema, still new granulation. but now, 
cellularity markedly decreases. little infl infiltrate. minimal mphage 
and neuts. edema goes away. and vascularity decreases. some vessels 
thrombose and die off. we keep only the vessels we need to keep the 
tissue viable. but one thing INCREASES a lot. the fibroblasts make a LOT 
of collagen. end stage of healed wound with lots of repair of damage 
tissue is marked by a lot of collagen - type III first, then remodelled 
to type I. so will look like grey, contracted, not-that-vascular area, 
consisting of collagen aka fibrous CT.

now, what about that large gap in the surface epithelium? do we actually 
close that, or what? 
well, while blood/fibrin clot is there, the surface is just dry - there 
is a scab filling in the defect in the surface epithelium. this is dried 
blood, fibrin, inflammatory cells caught in there. this is an 
inhospitable environment for cells. epi cells can't grow into it or over 
it. they can only try to grow along granulation tissue that will support 
their existence. but here we are talking about a deep wound. the 
epithelium may try to grow under the clot, along the granulation bed, but 
that doesn't work. so there is a lot of mitotic activity at the edges of 
the surface epi, but no real growth. but, when granulation bed fills in 
most of the defect, such that the granulation tissue is up level with the 
surface epi, the surface epi will grow across it.  the surface epithelium 
cannot regenerate several structures in this situation - the adnexal 
structures. the adnexal structures are what? things like hair follicles, 
sweat glands, and sebaceous glands. these structures can't be reformed, 
and why? because the stem cells are missing. for these structures to be 
formed, you need a remnant of them, and those are already lost. this is 
why wound surfaces do not grow hair. 

so, when you get this secondary union, most of blood clot is removed by 
mphages, granulation fills in, and you end up w/avascular area of fibrous 
CT and collagen. 

how will this wound look in two or three mos? what happens to this area? 
well, it will reduce itself in size by about 90%. the degree of 
contraction in a wound is phenomenal. the reason it contracts is several 
reasons. we have decreased cellularity and edema for one. but also, the 
fibroblasts are contractile, and they contract! they become less active. 
the collagen gets remodelled into type I and reduced, and more densely 
packed...so the wound gets much much smaller. 

having said that...
get back to the various stimuli for fibroblasts and endothelial cells. 
the stimuli for fibroblast growth and directional movement of cells is 
based on two things. one, the ability to attach to things like 
fibronectin and laminin - so ECM plays big role in triggering motility 
and differentiation. but other factors which trigger them are humoral 
components and growth factors in here. in terms of fibroblasts, the 
growth factors needed are similar to those in chronic inflammation - 
mainly, PDGF platelet derived growth factor, from platelets and 
fibroblasts; FGF, fibroblast growth factor; TGFb transforming growth 
factor beta, which is mitotic in low concentrations and inhibits mitotic 
activity in high concentrations, and is very fibrogenic; TNF tumor 
necrosis factor, which comes from macrophages. so in a plain chronic 
inflammatory rxn we have these same factors stimulating fibroblasts. what 
about endothelial cells? they are triggered by FGF and by a vascular 
endothelial growth facter VEGF and by TGFb. remember, we also need 
attachment to the adhesive glycoproteins to allow for directional 
movement of the cells...so they grow in the right formations and stuff.

so, we talked right now about healing where repair process is occuring by 
replacement with fibrous CT. when you have an inflammatory process either 
in infarcted part of kidney, or after acute bronchopneumonia in lung, 
need to think about how that tissue heals, and how well the parenchymal 
cells can or can't regenerate to rebuild the destroyed tissue. can they 
or can't they? that can be difficult early on but hopefully we'll figure 
it out going through the homework problems (is there a handout for 
those?)

any questions?
if you could increase the vascularity or prevent some of the new vessels 
from shutting down, could you decrease scarring? well, the blood supply 
is needed for fibroblasts to grow and make collagen. main way to reduce 
degree of scarring, though, is by preventing the acute phase of 
inflammatory response after damage occurs, because that's the most 
damaging thing to the tissue. use of corticosteroids will decrease the 
acute inflammatory response - we use it for inflammation of brain, eye, 
joint, to prevent fibroplasia, tissue destruction, and scarring. but in 
other areas eg skin, bone healing, etc, we don't want to retard 
inflammatory response because it aids healing process. overall, 
inflammatory response is beneficial, b/c it brings in all these factors 
and componenets, except in areas where you can't regenerate.

why doesn't fetal tissue scar?
more stem cells that can regenerate, lots of remodelling going on anyway.


before we stop, a couple of names on the board...
start thinking about differences between these things even though not all 
on exam.

granulation tissue: know what it is, and how it differs from granuloma
repair: know what it is and how it differs from regeneration
fibrinous: know what it means, and how it differs from fibrous

note that the 48-96 hr clot is fibrinous, and the final scar is fibrous.
fibrin represents polymerized fibrinogen. fibrinogen comes from blood 
(liver). 

resolution: know what it is. basically it is complete restructuring of 
damaged tissue to normal tissue. you have inflammatory response resolving 
into preexisting normal structure.

organization: know what it is. when people talk about infarct healing, 
they say it "organizes" or they say tissue becomes organized. they mean 
it has undergone repair and developed a lot of scar tissue.

---break---

slides

* what looks to me like an alfalfa cube sitting inside the lumen of an 
intestine that has been opened up. oh, it's a corn cob. 

* nice scenic slide of mountain and trees. when you take pictures when 
you are inebriated, mountains look upside down, he says.

* back to the nasty stuff. fibronectin molecule - remember it first comes 
from plasma, then is made by mesenchymal cells. is large molecule that 
has well defined portions with which it can attach to other things eg 
fibrin, collagen, heparin, integrin molecules, etc. so fibroblasts and 
endothelial cells can attach to the RGD section of fibronectin via the 
fibronectin integrin binding site (RGD section). this molecule important 
for motility and further differentiation of the cells.

laminin allows cell binding in one region. remember it is in basement 
membranes. the endothelial cells bind to the cell binding site. other 
parts of molecule bind to other ECM components. so by virtue of binding 
to matrix and to cells, allows differential movement of cells 
andmotility.

* granulation tissue on dorsum of dog. surface skin has been removed. 
it's all neatly debrided. dog was barking and neighbor through acid on 
it. that's why it is such a "clean" looking wound. necrotic material was 
washed off and the underlying tissue is granular, unevenly surfaced, 
intensly hyperemic, moist, edematous, and bleeds readily. this photo is 
after 5-6 days. very good healing taking place. not much in terms of pus 
or debris on surface. surface epi will have a hard time crossing the gap. 
ultimately this will contract by 90%.

* histology of granulation tissue. very surface: background eosinophilic 
material which seems very amorphous and unstructured. mixture of 
inflammatory cells - some are neutrophils, some are monocytes, some are 
macrophages. they are in a bath of proteinaceous edema fluid.
beneath that, are some really good macrophages, with vacuoles in 
cytoplasm, phagocytosed material in them, eccentric nuclei. also other 
mononuclear cells.
deep to that we find some blood vessels, some macrophages, some 
neutrophils
deep to that, more organized granulation tissue- lots of new blood 
vessels which we recognize as newly formed bvs mainly b/c a preexisting 
bv would have very flat, unremarkable endothelium, and these have rounder 
endothelial cells, larger and more prominent andmore densely packed, with 
occasional mitotic figures. what keeps these structures open is they are 
filled with blood. around them is newly formed ECM, and a lot of 
inflammatory cells. just as we'd expect. these bvs can't go any further 
until debris has been removed by mphages.  with special stain, we can see 
that near the wound sruface there is much less collagen, and there is 
more collagen near the area of the blood vessels.

macrophage: big cells. oval/eccentric nuclei. vacuolated cytoplasm.
neutrophils: smaller, w/segmented nuclei.

*lots of highly structured, eosinophilic material: collagen. this is deep 
in the wound. the big purple elongated cells are activated fibroblasts.  
sometimes they are kinda rounded, plumper. a couple of newly formed blood 
vessels are present.

*still more structured. much more collagen, more fibrillar, the 
cellularity has markedly decreased, almost no inflammatory cells. the 
vascularity has markedly decreased. what's more prominent is the CT. the 
fibroblasts are becoming more quiescent. they are flatter or more oblong, 
decreased in number.  with special stain we can see how very much 
collagen (pale blue stuff) is there. the spaces are blood vessels.

*wound surface....whole top thing is scab: dead/dying neutrophils and 
dried fluid and fibrin and stuff. at bottom, is all granulation tissue. 
can see long tongue of epithelial cells trying to grow across granulation 
tissue under the scab. close up can see it much better. note that there 
are lots of blood vessels growing in the granulation tissue, and there's 
a lot of collagen - active fibrovascular tissue - and the epithelium 
trying to cross it. 

after healing has taken place and you have restitution of the surface 
epithelium, notice that there are no hair follicles or glands in that 
part of the skin.

*in some organs when major damage occurs, how does tissue restructure? 
myocardium - myocardial muscle here is not normal. the nuclei are gone. 
it is necrotic muscle. there has been massive infarction here. you can 
see some areas are totally replaced with lighter staining fibrous CT. if 
you have an infarct in heart, dead muscle must all be removed by mphages 
and while that is going on, the infiltrating granulation tissue will 
replace the area of damage. the only way to fix this is repair by 
granulation tissue and scarring. you end up without functional muscle in 
that zone, because heart can't regenerate.

*normal cardiac muscle: nice centrallly located  nuclei, striated muscle
*granulation tissue with necrotic muscle remnants. there are numerous new 
blood vessels present. 

*another slide of the same thing. dead heart muscle, no nuclei visible. 
striations are gone. b/c of structured outline, ghostlike appearance, we 
know this is coagulation necrosis aka ischemic necrosis. 

*what happens when you have peritonitis. a lot of fibrinopurulant exudate 
on surface of intestinal tract, eg, covering the serosa. to have healing 
take place in abdomen...say dog ruptured lg intestine, and as it 
recovers, what happens to the fibrinopurulent exudate that's covering the 
intestinal tract? how long does it take to go away?  here on this slide 
we see a lot of neutrophils. some are dying. the nonstructured 
eosinophilic background material is proteinaceous fluid and fibrin. as 
you approach the serosal lining of the intestinal tract, we see an 
intense cellularity. the exudate on the serosa is an irritant. we start 
seeing an outgrowth of fibroblasts, newly occuring blood vessels, etc. we 
see formation of granulation tissue occuring where the fibrinopurulent 
exudate is. if it takes more than 4-6 days for exudate to go away, we end 
up with fibrous CT covering the serosa. if you have two intestinal loops 
bathed in this exudate, you can end up with fibrous adhesions between 
them. as fibrous CT remodels and contracts, you can get marked 
constrictions of lumen, and potential intussusceptions, decreased 
peristalsis, increased probability of torsions, etc. severe abdominal 
injuries can cause this. these adhesions can be a very serious problem. 
whenever you have a fibrinopurulent exudate, you worry how long is it 
there, how long will it take to remove, and what happens if it takes over 
a week or two.

slide: serosa with granulation tissue on it. it's growing into the area 
of the exudate. deeper in the serosa you can see the newly forming 
vessels, and on the surface of serosa we see the mesothelial lining cells 
which are no longer flat, but are rather large, look like macrophages. 
they undergo hyperplasia during inflammatory response. beneath them we 
see the inflammatory reaction.

*what happens in kidney when you have destruction of tubular cells via a 
nephrotoxin? here we see a tubule with no lining cells left. all the 
material inside is just necrotic epithelium. can that repair? well. we 
should think about it until monday. note in adjacent tubule, there are 
some epithelial cells that are very dark, with prominent nucleoli, and 
mitotic figures. so this tubule has actively growing epithelial cells. 
are they regenerating something? how effectively can they do this? what 
has to be in place in order for them to totally restructure the tubule?

another kidney damaged by nephrotoxin. one totally necrotic tubule. 
adjacent tubule has a lot of mitosis going on. the epithelial cells are 
very large, w/prominent nuclei and a lot of hypoplasia. can this work? 
will it function?
another picture of the same thing. learn to notice mitotic figures. 

so, til monday, try to work out these problems from the handout he gave 
us. especially the problem you are assigned to!!!

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