Studies on the histochemical begavior of connective tissue elements following injury
With the emergence of the concept of cellular activity and dominance in biological reactivity, the functional significance of connective tissue was lost sight of and the concept of connective tissue as an integrated system is of recent acceptance. Although there have been numbers of investigators who have called attention to the alterations seen in connective tissue elements in various disease states, it was not until 1942 when Klemperer and his associates published their classic paper on "diffuse Collagen Diseases" that a new era on the field of
connective tissue research has been opened. Since that time, many volumes of pertinent data have been accumulated concerning connective tissue in the world medical literatures.
Despite such an intensive investigations on various aspects of connective tissue, there are still many doubtful and unexplored fields particularly on the matter of morphogenesis. furthermore not all the informations available confirm to each other but in many instances they are even contradictory.
The fibrillar structures in the interstitial spaces are generally composed of three types of fibers, namely, collagen, elastic fiber and reticulum fiber. There are some who believe that the fibers were shed from the superficial protoplasm of
the fibroblast and incorporated directly into tissue as collagen. Others have held that collagen is precipitated from the ground substance without any need for cellular activity. The formation of elastic fiber is not understood. The formation of ground substance presents even greater problems than collagen fibers. The relationship of reticulum to collagen is a matter of intense discussion.
The purpose of this study is to characterize various connective tissue elements on the basis of the more detailed and precise tinctorial criteria in the hope that inaccuracies, misunderstandings and much confusion presently prevailing in this
field could be diminished.
It is held that wound healing recapitulates the events of connective tissue growth. Therefore a large rectangular defect was made in the flank of rabbits to follow the events taking place in the newly growing connective tissue. At the same time, ascorbic acid and adrenalin were given to elucidate some of the mechanisms involved in their influence on connective tissue growth. An additional observation was made on a certain human diseases where fibrosis or cicatrization commonly occurs.
MATERIALS AND METHODS
1. Experimental animals
Healthy adult albino rabbits weighing around 2.0kg were used. The animals were fed and caged in the same manner for 2 weeks before the experimental procedures. The animals were divided into the following groups.
A. Normal group ………………………………………………………………20 rabbits
B. Administration of adrenalin ………………………………………… 20 rabbits
C. Cortisone administration ………………………………………………10 rabbits
D. Vitamin C administration ………………………………………………10 rabbits
E. adrenalin and vitamin C administration ……………………………10 rabbits
F. Adrenalin and Cortisone administration ……………………………10 rabbits
G. Cortisone and Vitamin C administration…………………………… 10 rabbits
H. Adrenalin, Cortisone, and Vitamin C administration ……………10 rabbits
Before wounding, tattoo is made around the expected margins of the wound at the 0.5cm. intervals. This was to access the gross features of wound contraction. Each animals was subjected to make 5.0×5.0cm rectangular deep skin wound extending up to the fascia of the abdominal muscles. Immediately before and each day afterwards, gross photographs of the wound in an identical condition was made. Measurements of three aspects of the wound were made every day accordingly.
Administration of the drugs designated was made starting from 7 days ahead of wounding until the end of experiment. The drugs were given in the doses of 0.1cc per kg/BW(adrenalin), 25.0mg per kg/BW(cortisone), 25.0mg per kg/BW(ascorbic acid), respectively.
After wounding the animals were killed in the order of 1, 3, 5, 7, 9, 12, 15, 18, 20 and 30 days. The areas of wound was entirely resected and fixed into the Lilie's alcoholic formalin and 10% buffered neutral formalin. Tissue blocks were embedded in paraffin and sections were serially prepared.
3. Human materials
As a representative of non-specific inflammation and fibrosis, acute and subacute appendicitis and fibrous obliteration of appendiceal lumen; acute and chronic cholecystitis were selected. As a representative of specific granulomatous inflammation, cases of pulmonary tuberculosis were selected. There are a certain
carcinomas which are frequently accompanied by a marked desmoplastic reaction.
Cases of this variety were also chosen for the study.
The materials all came from the surgical and autopsy files of the department of pathology of Yonsei University College of medicine. Although the number of cases examined was as follows (appendix 779; gallbladder 123; lung 249; carcinoma 20), 10
cases from each variety were serially sectioned and subjected to the following histochemical procedures.
4. Histochemical stains utilized
a. Hematoxylin & Eosin stain
b. Periodic acid schiff reaction (P.A.S.)
c. Gomori's Aldehyde Fuchsin stain
d. Verhoff's Elastic fiber stain
e. Toluidine blue stain
f. Phosphotungstic Acid Hematoxylin stain (P.T.A.H.)
g. van gieson stain
h. Heidenheim's Iron Hematoxylin stain
I. Wilder's reticulum stain
j. Rinehart & Abul-Haj's Colloidal Iron stain.
A. Findings in experimental animals
1. All throughout the experimental period, the animals in each group showed no significant weight changes and they survived the expected experimental period.
2. A comparison of the measurements made on the contraction of wound showed that the administration of cortisone, cortisone and adrenalin, cortisone and vitamin C all caused delay as compared to the normal group. The other groups of animals more or less similar rate of contraction as the normal group.
3. Histochemical changes
a. Normal group: The early stage is principally represented by reaction to injury, onset of necrosis and exudate accumulation; a coagulum is formed of blood, serum on the surface and edges of the wound. The cells at this stage are those of acute inflammatory cells. The capillaries proliferate at this time. The
histochemical changes may be summarized as the following. At this stage the colloidal iron, toluidine blue and P.A.S. stains all are positive. Particularly there is an early progressive increase of acid mucopolysaccharides stained by colloidal iron which begins to stain slightly immediately after wounding. By 5th to
9th day period the intensity of the staining reaches its maximum. The intensity of metachromasia by toluidine blue also progressively increases but slightly less than colloidal iron. The P.A.S. reaction is less reactive than the formers but moderate intensity is noted after 9 days. van Gieson and P.T.A.H. stains demonstrate appearance of thin newly formed collagen fibers as early as 7th day.
At this stage, there is no demonstrable elastic fiber. The pattern of reticulum fiber were interesting in that the appearance as demonstrable by Wilder's and P.T.A.H. stains was quite prominent at an early stage. These stains showed fine
argyrophilic fibers of fibroblasts as well as thin fibrin strands which appeared to contact each other. Some have actually occupied the same location. Argyrophilic fibers became thicker and thicker after 7 days and some transformed into red collagen fibers.
Coinciding with the rapid contraction of wound there occurs a marked and progressive proliferation of fibroblasts and deposition of collagen fibers. The newly formed collagen fibers in the beginning are very thin and delicate and stain rather weakly by the van Gieson. As the time ;apses these become thicker and stain heavily with van gieson, P.T.A.H. or P.A.S. Colloidal iron stain demonstrated that the staining reaction of acid mucopolysaccharides rapidly decrease with the appearance of collagen. The reticulum stain also shows that the argyrophilic fibers gradually transform into collagen more frequently at this stage. The elastic fibers are formed but the number is very scarce and its appearance quite late. However the elastic fibers at the neighboring intact skin margins stain heavily. These are
twisted, wrinkled and duplicated. Fine and thick fibers form masses of elastic tissue at the wound margins. The elastic membranes and fibers in and around the blood vessels also show thickening and fragmentation. Some of these thick elastic membranes behaved tinctorially different from the normal internal elastic membrane of blood vessels by P.T.A.H. and colloidal iron stains.
b. adrenalin group: The histological and histochemical changes seen in this group of animals did not differ greatly as compared to the normal. The regular sequences of connective growth was observe. However there was a less intense staining of acid mucopolysaccharides and slightly less activity of fibroblastic proliferation. The acid mucopolysaccharides stained slightly quite late but the collagen formation was slightly hindered at the last stage. Therefore the late stage of fibrogenesis was
slightly hindered in this group. The elastic fiber and reticulum fiber were not significantly different from that of the normal.
c. Cortisone group: The most significant finding in this group was a remarkable reduction in the staining reaction of acid mucopolysaccharides in the initial period. The vascular and cellular reaction were also reduced. The formation of collagen fiber was seen but the intensity and its thickness were not comparable to the normal. The changes in the elastic fiber or reticulum fiber from the normal were not appreciable.
d. vitamin C group: The changes seen in this group did not materially differ from that seen in the normal group. The staining patterns of various histochemical methods were similar to the normal.
e. Other groups: The general pattern of connective tissue growth in the remaining groups was as follows. The administration of adrenalin and Vitamin C together showed pattern almost identical to the normal. Cortisone and Vitamin C, adrenalin
and cortisone, Adrenalin, Cortisone, and Vitamin C groups all caused changes similar but less than that seen in the cortisone group.
B. Findings in human diseases
1. Non-specific inflammation
The changes in acute suppurative appendicitis and cholecystitis showed the changes very similar to the initial stages of the experimental wound healing. since the materials selected did not give us any specific date of onset the chronological stud was not possible.
The appearance of thin delicate collagen fiber as demonstrated by van Gieson stain was seen in subacute type of appendicitis. The reticulum fiber was exceedingly abundant in layers of appendix. The appendices obliterated by dense collagen fibers showed no accumulation of acid mucopolysaccharides. In acute stages however there were positive colloidal iron stains around the blood vessels. Both in gallbladder and appendix, an extremely interesting behavior of elastic fiber was noted. The submucosal and muscular layers of the organs were heavily stained with
both aldehyde fuchsin and Verhoff's stains. The increase was not only observed in these areas but the walls of the blood vessels also showed similar changes. Iron hematoxylin, colloidal iron and P.T.A.H. including P.A.S. all stain membranes of elastic fibers in the intima of blood vessels. The observed increase in elastic
fiber as demonstrated by the usually accepted elastic stain behaved quite differently after P.T.A.H. and colloidal iron stains. In other words, the normal elastic membrane staining of P.T.A.H. was different from the staining patterns of these abnormally increased elastic fibers. It was also interesting that
fragmentation and increase of elastic fiber were also very closely accompanied by staining of acid mucopolysaccharides.
2. Specific granulomatous inflammation
Various stages of granuloma formation in Jung tuberculosis were examined. In the majority the examined cases all showed healing stages of the disease although there were active caseation necrosis in areas. As far as collagen fiber formation is concerned, there was no significant difference from the other forms of inflammatory reparative process but the reticulum fiber in transforming into mature collagen was most evidently demonstrated in these granulomas. However there were reticulum
fibers that were not participating in the collagen fibrogenesis. The hyaline connective tissue gave very intense reaction with Van Gieson and P.A.S. but the peripheral portions of the cavity showed thin delicate collagen fibers. Acid mucopolysaccharides was not stained in any granulomas. The most interesting finding
was seen again in the elastic fibers. although the granulomatous process may have caused fragmentation but the increase in abnormal form of degenerated elastic fibers was not only seen in and around the blood vessels but also in the relatively uninvolved pulmonary parenchyma. Again some of them behaved variably to P.T.A.H. and colloidal iron stains.
3. Desmoplastic reaction in carcinomas
Histochemical characteristics of the collagen fibers and of elastic fiber and reticulum fiber were not different from the other human materials. However, the collagen fibers were basically of two types, one in which intense P.A.S. reaction
is observed and the other staining less intensely. Acid mucopolysaccharides were sometimes associated with deposition of reticulum fibers in some lesions. However the most striking finding here again was the enormous increase in elastic fibers.
These fibers gave varying staining patterns somewhat different from the normal pattern.
COMMENT AND CONCLUSION
As mentioned earlier there are two schools of thoughts on the nature and formation of collagen fibers. From the histochemical data obtained through the present investigation, it seems reasonable the both cellular activity and acid mucopolysaccharides play essential role in collagen formation. Accumulation of acid mucopolysaccharides during the initial stage and disappearance of this material when collagen fiber begins to form suggests that somehow acid mucopolysacchrides are used during the collagen synthesis. On the other hand, the source of acid mucopolysaccharides seen in this stage must be elucidated. Grossfeld et al. have definitely established that fibroblasts are capable of producing acid mucopolysaccharides in tissue cultures. asboe-Hansen believe that mast cells secret hyaluronic acid. It could also derive from circulation. The studies of this investigation revealed that acid mucopolysaccharides were seen both around th blood vessels and within the cytoplasm of fibroblasts. Therefore the conclusion that the cells and the ground substance both as responsible factor in collagen fiber
formation is reached.
The behavior of elastic fiber during the morphogenesis of connective tissue was particularly interesting in the neighboring tissue rather than within the granulation tissue newly formed. The formation of elastic fiber was extremely late and the number was scarce. However abnormal proliferation of elastic tissue occurred at the margins of wound and in the interstitial tissues of the human diseases. Although the morphogenesis of elastic fiber could not be revealed through the study, the identification of these abnormal forms of elastic fiber is quite significant. Tinctorial reactions carried out tend to show that these were somewhat different from the normal elastic fiber and that these may be derived from degenerated collagen or as a result of disturbances in the metabolic processes normally leading to collagen or reticulum fiber deposition in connective tissue.
The reticulum fibers are not only formed by the cytoplasmic extensions of fiberoblasts but also from precipitated fibrin and the interstitial fibrillar components. Not all the reticulum fibers transform into collagen but some are just in contact with the collagen fiber. from the data obtained above, it is concluded
1. Contraction of skin wounds is greatly influenced by the histochemical patterns of the granulation tissue inside the wound.
2. The histochemical reaction of acid mucopolysaccharides during the morphogenesis of collagen fiber has a definite relationship to the deposition of collagen fiber.
3. Formation of collagen fiber needs both cellular and interstitial components.
4. Morphogenesis of elastic fiber is not clear but in reacting connective tissue there occurs abnormal form of elastic fiber which may be derived from degenerated collagen or as a result of metabolic disturbances in reticulum or collagen fiber formation.
5. Reticulum fibers contribute significantly to the formation of collagen but there are other which do not participate in this. The reticulum fiber appears to be derived not only from fibroblasts but from the fibrinous precipitates in the interstitial tissue.
6. adrenalin has an inhibitory action of the acid mucopolysaccharides and delay in fibrogenesis. Hyperglycemia secondary to adrenalin injection must have been at
7. Cortisone causes a marked reduction in acid mucopolysaccharides in addition to inhibitory action on cellular and vascular reaction.
8. Vitamin C showed no significant histochemical changes in normal wound healing.
9. The human diseases showed seemingly same pattern as the animal. The abnormal elastic fiber was the most prominent.