Semmelweis University of Budapest. Department of Anatomy, Histology and Embryology. Béla Ajtai, M.D. COMPARATIVE MORPHOLOGY OF REACTIVE GLIOSIS

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1 Semmelweis University of Budapest Department of Anatomy, Histology and Embryology Béla Ajtai, M.D. COMPARATIVE MORPHOLOGY OF REACTIVE GLIOSIS Theses for PhD degree (short form) Tutor: Dr Mihály Kálmán, M.D., Ph.D. Budapest, 2002.

2 2 Reactive gliosis is a characteristic phenomenon that follows the different types of central nervous system (CNS) insults. It is to be considered as the reaction of the neural tissue where the predominant role is played by the astroglia. The reactive gliosis has been blamed for a long time for the failure of neural pathway regeneration after injury. In the early stages of development, regeneration still may occur after injury, however, in this case no reactive gliosis is formed and, in fact, the astroglia seem to play an essential role in maintaining and supporting the nerve fiber growth. The molecular changes leading to inhibition of nerve fiber growth after injury are not completely understood at this point, therefore it is very desirable to develop an experimental model where the spatial and temporal occurrence and course of fiber growth inhibition are well known. Another question of utmost importance is, how does the appearance of reactive gliosis and the loss of regeneration fit into the course of neural tissue development and maturation. What is the biological reason to have these phenomena? These questions are best answered by in situ investigations of lesions to the brain at different stages of its development. In my investigations I looked for an answer to these questions by investigating the reactive gliosis following mechanical lesions to the brain. 1. The formation of reactive gliosis in the developing rat brain The capacity of the neural tissue to form an inhibitory reactive gliosis appears at some time during the neural histogenesis. Our goal was to identify the earliest developmental stage at which a reactive gliosis similar to that in the adult animal may be formed. Since the development of the cerebral cortex and the subcortical structures has an asynchronous schedule, the question might arise, whether the appearance of the reactive gliosis depends on general (e.g. immunological) or rather local tissue factors. To explore this hypothesis, we compared the temporal profile of the formation of reactive gliosis in two regions of the brain: in the cerebral cortex and in the diencephalon. These regions can be injured simultaneously, in one operation making the design of experimental setting and the analysis of results a whole lot easier. In the first phase of the experiments we performed simple, light stab wound lesions. The results we got have suggested that during development the formation of the reactive gliosis might also be influenced by the severity of the injury. To analyze this, in further experiments we also compared the effects of light (stab wound) and severe (large stab along with suctioning to remove tissue) lesions. These lesions of different severity were performed at different pre- and postnatal stages, the results were analyzed after short as well as longer survival periods. 2. The relationship of reactive gliosis and nerve fiber growth The general opinion about the effects of reactive gliosis is gradually changing in these days. While earlier it was considered to be harmful to neural regeneration beyond any doubt, and every attempt was made to find a way to prevent its formation, nowadays more and more investigators think that it may have beneficial features as well, in fact some people think that it has a primary neuroprotective and fiber growth-enhancing role.

3 3 This apparent controversy of opinions makes it necessary to further analyze the relationship of reactive gliosis and nerve fiber growth at different ages, in different cerebral regions. Although the general opinion about the harmful effects of reactive gliosis to the loss of regeneration is still being held, the question arises, whether during development the appearance of reactive gliosis really happens at the same time as the cessation of regenerative capacity. We investigated the relationship of reactive gliosis and nerve fiber growth in three experimental settings: i, following incision of the site of the corpus callosum, starting at the 17 th embryonic day (before the appearance of the first, so-called pioneer fibers); ii, based on these results we expanded our investigations to callosal fibers that grew along alternative pathways while trying to get around the injury; iii, we also studied the features of nerve fiber growth following incision to subcortical structures (e. g. diencephalons). In these experimental models we attempted to identify the exact time and developmental stage, at which the regenerative capacity disappears. This might be a good start to further studies in which we would analyze the distribution of the factors regulating nerve fiber growth (e.g. components of the extracellular matrix, cellular adhesion molecules, growth factors). However, this latter effort is only in the initial experimental phase at this point. 3. Comparative morphology of the reactive gliosis in brain regions with special astroglial structure The experimental data piled up so far about the reactive gliosis were almost exclusively obtained from those regions of the brain, where the astroglial processes do not have an exquisite unidirectional arrangement. It would be quite interesting to compare the reactive gliosis of these regions with that of those where the astroglial processes are specially arranged and of those where the typical glial cell is ependymoglia. The question arises, whether an astroglial process system that is originally directed to one specific direction has the capacity to rearrange these processes to form a demarcation at the lesion site. Examples of these special glial populations are the Bergmann glia (the reaction of which we studied after stab wounds to the adult rat and chicken cerebellum) and the ependymoglia of the optic tectum in fish (goldfish in our experiments). It is a well known phenomenon in mammals and birds that those brain regions that normally are not (or just barely) immunopositive to GFAP (glial fibrillary acidic protein) will respond to an injury with a GFAP-immunpositive reactive gliosis. The brains of animals with a predominance of ependymoglia have only a few GFAPimmunonegative regions, the glial reaction of which has not been studied yet. To investigate these regions, we performed stab wound lesions to the lobe of the vagal nerve to compare its glial reaction to those seen in the also GFAP- immunonegative regions of mammals and birds.

4 The experiments were conducted with strict observance of the rules set by the Animal Experiments Code of Ethics (anesthesia of proper depth and duration, adequate postoperative care, etc.). To perform a lesion in the newborn or early postnatal rat brain, the skin of the head was incised, pulled sideways and then the skull incision was made. The various kind of lesions were performed through this skull incision. For the studying of the appearance of reactive gliosis we used: i, simple stab wound with a thin needle; ii, stabbing and suctioning with a thick needle attached to a water jet pump. To study the relationship of reactive gliosis and neural fiber growth we performed incisions to the cerebral cortex, corpus callosum and diencephalons using a sterile razor blade fragment. In the prenatal age (rat embryos) these lesions were performed using our special intrauterine technique, after the induction of proper anesthesia in the mother animal. To perform stab wound lesions in the adult rat and chicken cerebellum, the head of the animal was put in a stereotactic device. The skull was opened using a dental drilling machine and subsequently we made a dural incision. We then inserted a thick sterile needle into the cerebellum through all its layers. The lesio ns of the goldfish brain were performed by stabbing the needle through the skin and skull. After different periods of survival the animals were overanesthetized by a gradually escalating dose of the anesthetics. The brains of the goldfish and newborn rats were fixated by immersion only, in all other cases we applied perfusion fixation using a 4% paraformaldehyde solution. This was generally followed by immersion fixation for additional 2-3 days in the same kind of fixative. The brains were then sectioned using a vibrating microtome (Vibratom). The slices processed for electron microscopy were embedded in Durkupan then semithin sections were obtained where the area of interest was identified. Our most frequently used tissue staining method was based on the immunohistochemical detection of various molecules. The two most commonly used molecule was the GFAP (glial fibrillary acidic protein, to investigate the reactive gliosis) and the NFP (neurofilament protein, to stain the nerve fibers). Additional immunohistochemical reactions were also performed against other glial intermediate filaments (vimentin, nestin), and other molecules that regulate nerve fiber growth (laminin, fibronectin, chondroitin sulfate, transforming growth factor beta, pleiotrophin, L1, tenascin, neural cell adhesion molecule). The various steps of the immunohistochemical reaction were separated by the washing of the floating sections in 0.1 M phosphate buffer, for a good 30 minutes. The sections were treated with a 3% solution of hydrogen peroxide, followed by a 90-minutelong incubation in 20% normal goat serum to block any nonspecific antigen binding. The sections were then incubated in the solution of the primary antibody, on 4 C for 40 hours. In the next step the slices were incubated in the solution of biotinylated secondary antibodies raised against the above primaries, for 90 minutes. The sections were then put into a 1:100 solution of streptavidin-biotin-horseradish peroxidase complex for 90 minutes. The immunocomplexes were visualized by the diaminobenzidine (DAB) reaction, on room temperature. The stained slices were then mounted on glass slides and after proper drying (approximately 24 hours) they were covered with plastic covers using DePeX. For certain brain regions Nissl staining was used. 4

5 5 In the rat cerebral cortex on or before the 20 th embryonic day neither the light stab wounds, nor the severe injuries (even those resulting in permanent deformity or strange tissue impaction) have led to formation of reactive gliosis. The light stab wound healed without residua, the severe ones resulted in formation of cortical invaginations or porencephalia. In cases of invaginations the cortical structure (arrangement of layers, vascular structure) appeared to be modified according to the deformity. The surface was covered by a glia limitans, the structure of which was similar to that of the intact cortex. The capacity of the astroglia in the rat cerebral cortex to respond to an injury (the so-called glial reactivity) becomes similar to that of the mature animal after the 5 th postnatal day, therefore lesions at or after this age were regularly followed by permanent reactive gliosis, with no regards to the lesion severity. Surveying the literature data on rat cerebral cortical development we concluded that the appearance of cortical glial reactivity can be linked to cessation of neuronal migration and the transformation of the radial glia to astrocytes. The lesions do not provoke an earlier radial glia-astrocyte transformation and the radial glia do not become GFAP-immunopositive. Between the day of birth and the 5 th postnatal day the appearance of the reactive gliosis depends on the severity of the lesion. If the injury is mild and heals before the appearance of the glial reactivity (i.e. 5 th postnatal day), no reactive gliosis will form. In those cases however, where the effects of the damage persist up to the 5 th postnatal day, reactive gliosis will appear. According to this, if severe enough, even injuries to the newborn brain may provoke reactive gliosis. Another very important observation of ours is that gliosisprovoking lesions, performed in the period between the 1 st and 5 th postnatal days, will result in reactive gliosis uniformly on the 7 th -8 th postnatal day, therefore the earlier the injury occurs, the longer is the latency of the onset of the reactive gliosis. Thus we can conclude that in the early postnatal period, it is the appearance of the reactive gliosis that can be linked to a certain age (in the rat cortex the 7 th -8 th postnatal day), not its elicitability. This might explain the apparent controversy of literature data on the appearance of the reactive gliosis, as those investigators, instead of looking for the earliest age at which the reactive gliosis actually appears, were rather trying to find the earliest age at which performed a lesion will result in reactive gliosis. As we conclude from our results, this latter age limit in the cortex can vary considerably according to the severity of the lesion. Contrary to that seen in the cortex, lesions to the developing rat diencephalon have resulted in permanent reactive gliosis also when performed in the embryonic age (on the 18 th embryonic day or later). The elicitability of the reactive gliosis can therefore be dated to an earlier phase of development than generally accepted in the literature. This also supports or hypothesis that age of the appearance of reactive gliosis is different in the various brain regions. The likely explanation of this is the fact that the appearance of glial reaction requires a certain level of tissue maturity and the various brain regions develop and mature asynchronously (the subcortical regions preceding the cortex). This observation is the other explanation for the variability of literature data on the appearance of the reactive gliosis. Fitting the appearance of the reactive gliosis to the sequence of developmental events enables us to adapt our observations to the human brain as well. Following the incisions through the corpus callosum we observed that lesions performed on the 17 th embryonic day never, the ones performed after the 20 th embryonic day always disrupt the development of the tract. If the lesion was performed before the

6 2 nd postnatal day, no reactive gliosis was formed at the site of fiber disruption, indicating that factors other than the reactive gliosis might be responsible for the failure of fiber growth/regrowth. We hypothesize that probably the damage of pioneer fibers and/or of the guiding glial populations is the cause. Besides these phenomena, in cases when the lesion was performed in the newborn brain or earlier, we also observed the formation of an heterotopic group of cells. This cell group was well demonstrable by Nissl staining. We think that as a result of the lesion the migration of these cells to the cortex was arrested and their differentiation was also disturbed. The heterotopic cell group was frequently located at the level of the corpus callosum and the disrupted fibers were not able to pass through its territory. The exact mechanism of this inhibition is yet unknown to us, the few growth-regulating factors we tested have failed so far to provide an explanation. Although the disrupted callosal fibers (injured between the 18 th embryonic and the first postnatal day) were unable to cross the site of the lesion, they grew well on alternative routes in the substance of the adjacent developing cortex. Along the course of these fibers we also observed the appearance of GFAP- immunopositive astrocytes, although at these age these were not present in other areas of the cortex. We think that the growing fibers are inducing the appearance of these astrocytes and conversely these astrocytes would support the fiber growth. Such phenomenon was suspected to exist earlier in in vitro studies but it has not been demonstrated in vivo yet, to our knowledge. At this age in the cortex it is the fiber ingrowth that seems to induce the appearance of GFAP-astrocytes, not the tissue damage. Alternative explanation is that these astrocytes were activated to interfere with the fiber growth but this effect can not manifest for some reason. Following transections of the corpus callosum, reactive gliosis similar to that of the adult appears only if the lesion was performed on the 2 nd postnatal day or later. The area of this reactive gliosis is not penetrable for the disrupted fibers. According to our results, therefore, it seems that in the developing rat brain the loss of regenerative capacity does not parallel the appearance of the reactive gliosis. The failure of regeneration before the onset of reactive gliosis is attributable to other, yet unknown factors. Besides the GFAP- immunohistochemistry we also used immunohistochemistry against nestin to study the reactive gliosis following corpus callosum transections. We observed that although the nestin immunostaining reveals the presence of astrocytes in the rat cerebral cortex as early as in the newborn animal, this method, just like the one for GFAP, indicates reactive gliosis only after lesions performed on the 2 nd postnatal day or later. Following lesions performed earlier than the 2 nd postnatal day, the immunostaining of nestin is also absent from the site of injury, just like GFAP, indicating the absence the astroglial population there. These observations prove that GFAP is a reliable marker for studying the earliest appearance of the reactive gliosis. Incisions to the developing diencephalon, in accordance with our previous results, were regularly followed by reactive gliosis, if the lesion was performed on the 18 th embryonic day or later. A very interesting observation of ours is that after lesions performed on the 5 th postnatal day or earlier, an aberrant nerve fiber bundle appears in the region of the reactive gliosis. We suppose that the growth of these fibers was induced by the reactive astrocytes, as such nerve fiber bundle was not present on the intact contralateral site. Searching the literature we did not find any demonstration of such 6

7 7 phenomenon. A significant difference from the one observed in the cortex is that the fiber growth in the diencephalons takes place along the lesion, not in the adjacent normal tissue. The relationship of reactive gliosis and nerve fiber growth varies therefore not only in one brain region at different stages of development but also in the different brain regions. This may explain why the issue of reactive gliosis and fiber growth is so controversial and debated in the literature. The morphology of the diencephalic/supportive and cortical/inhibitory reactive gliosis seemed to have the same morphology when studied by immunohistochemistry against GFAP, vimentin or nestin, therefore the morphological picture, at least on light microscopic level, does not indicate the function. The observed heterogeneities between the different brain regions and developmental stages indicate that the problem of regeneration will most likely not be solved by one general answer, but rather separate individual strategies may be needed in the different regions and developmental stages. Like in other areas of the brain, our investigations in the diencephalons about the fiber growth regulating factors have also failed so far to give any answers. It is a very significant achievement however that we were able to create both fiber growth supporting and inhibiting experimental models. Using these models for further biochemical and molecular biological analysis is very likely to provide new information about mechanisms of nerve fiber regeneration and reasons for its failure. Following stab wounds to brain regions with special astroglial structure, in the molecular layer of the cerebellum of the rat and domestic chicken we observed that the reactive gliosis is formed by the Bergmann glia, the astrocytes of the adjacent granular layer do not migrate to the site of the injury. The processes of the activated Bergmann glia remained parallel to the site of the lesion, they did not (or just barely) rearrange themselves. These results showed that from the three possible mechanisms of the origin of gliosis-forming cells (activation of the resident astroglial population, proliferation, or immigration from other brain regions), the first one (i.e. the activation of the local resident astroglia) seems to be the most important one, at least in the case of the cerebellum. Further electron microscopic studies are needed to understand how the most important goals of the reactive gliosis (formation of new surface, neuroprotection) are achieved in the apparent absence of demarcating glial scar. Although the processes of the Bergmann glia did not rearrange themselves after injury, the activated state of these cells was proven by the appearance and increase of their GFAP- immunopositivity. In rats the processes of the Bergmann glia are GFAPimmunopositive even in the intact cerebellum, but we could demonstrate the increase of this immunopositivity by using different dilutions of the primary (anti-gfap) antibody. A new observation is that the chicken Bergmann glia, which is GFAP- immunonegative under normal circumstances is also capable of expressing GFAP after injury. Investigating those regions of the goldfish brain that are devoid of GFAP immunopositivity we learned that -contrary to that seen in mammals and birds- no GFAP- immunopositivity appears in these regions, despite the injury. According to data in the literature, electronmicroscopic studies have shown that in these regions the glia contain only a fair amount of filaments and even the identification of these cells as astrocytes is highly doubtful. It is very well possible that these regions do not contain cells capable of GFAP expression or alternatively, the lesion does not induce GFAP expression.

8 8 Full papers on the topic of the theses List of publications Ajtai BM, Kállai L, Kálmán M (1997) Capability for reactive gliosis develops prenatally in the diencephalon but not in the cortex of rats. Exp Neurol 146: Ajtai BM, Kálmán M (1998) Glial fibrillary acidic protein expression but no glial demarcation follows the lesion in the molecular layer of cerebellum. Brain Res 802: Kálmán M, Ajtai BM (2000) Lesions do not provoke GFAP-expression in the GFAP- immunonegative areas of the teleost brain Ann Anat 182: Ajtai BM, Kálmán M (2000) Axon growth failure following corpus callosum lesions precedes glial reaction in perinatal rats Anat Embryol 202: Kálmán M, Ajtai BM, Sommernes JH (2000) Characteristics of glial reaction in the perinatal rat cortex: the effect of lesion size in the critical period. Neural Plasticity 7: Kálmán M, Ajtai BM (2001) A comparison of intermediate filament markers for presumptive astroglia in the developing rat neocortex: immunostaining against nestin reveals more detail, than GFAP or vimentin Int J Dev Neurosci, 19: Ajtai BM, Kálmán M. (2001) Reactive gliosis support and guide axon growth in the rat thalamus during the first postnatal week. A sharply timed transition from permissive to non-permissive stage. Int J Dev Neurosci 19: Full paper on other topic Kálmán M, Tuba A, Ajtai B (1995) Development of ependyma in neural transplants Int J Develop Neurosci 13:75-79

9 9 Citable abstracts on the topic of the theses Ajtai B, Kállai L, Székely AD, Kálmán M (1995) Reactive gliosis in fetal and neonatal rats. Eur J Neurosci Suppl 8:11 Ajtai B, Székely AD, Kálmán M (1994) NADPH-diaphorase positivity following lesions to the brain. Eur J Neurosci Suppl 7:53 Ajtai BM, Kállai L, Kálmán M (1996) Investigations on glial reactions to lesions in rat fetuses of different ages Neurobiology 4:288 Kállai L, Ajtai BM, Kálmán M (1996) Development of rat brain after fetal lesions. Eur J Neurosci Suppl 9:33 Kálmán M, Ajtai BM, Kállai L (1996) Capability of reactive gliosis develops prenatally in the diencephalon but not in the cortex. Soc Neurosci Abstr 22:1969 Ajtai BM, Kálmán M (1997) Glial reactions following stab wounds in the cerebellum and optic tectum of the developing and adult chicken brain. Neurobiology 5: Kálmán M, del Campo P, Sommernes JH, Ajtai BM (1997) Comparative study on reactive gliosis after stab wounds and partial cortical ablations in developing rats. Neurobiology 5: Kálmán M, Ajtai BM, Sommernes JH (1998) Characteristics of the formation of glial reaction in the perinatal rat brain. Neurobiology, 6: Ajtai BM, Kálmán M (1998) Effects of perinatal lesions on the development of the rat corpus callosum. Neurobiology, 6: Ajtai BM, Kálmán M (1998) Effects of perinatal lesions on the development of the rat corpus callosum. Eur J Neurosci Suppl 10:55 Ajtai BM, Kálmán M, Sommernes JH (1998) Characteristics of the formation of glial reaction in the perinatal rat brain.

10 10 Soc Neurosci Abstr 24:1795 Ajtai BM, Kálmán M (1999) Effects of late prenatal and neonatal lesions on the growth of the rat corpus callosum. Neurobiology 7: Ajtai BM, Kálmán M (1999) Nestin immunostaining in the lesioned prenatal cortex: astrocytes are present but not reactive. Soc Neurosci Abstr 25:790 Ajtai B, Kálmán M (2000) Effects of perinatal lesions on brain development-role of migration disturbances? Neurobiology 8: 288. Kálmán M, Ajtai B, Takács P (in press) Reactive glia promotes axon growth: a sharply timed transition of permissive stage to non-permissive stage. Neurobiology,