Astroglial Scar Formation Research Articles

PDGFR-β as a Positive Regulator of Tissue Repair in a Mouse Model of Focal Cerebral Ischemia

Although platelet-derived growth factors (PDGFs) and receptors (PDGFRs) are abundantly expressed in the central nervous system, their functions largely remain elusive. We investigated the role of PDGFR-β in tissue responses and functional recovery after photothrombolic middle cerebral artery occlusion (MCAO). In the normal adult mouse brain, PDGFR-β was mainly localized in neurons and in pericyte/vascular smooth muscle cells (PC/vSMCs). From 3 to 28 days after MCAO, postnatally induced systemic PDGFR-β knockout mice (Esr-KO) exhibited the delayed recovery of body weight and behavior, and larger infarction volume than controls. In Esr-KO, PC/vSMC coverage was decreased and vascular leakage of infused fluorescent-labeled albumin was extensive within the ischemic lesion, but not in the uninjured cerebral cortex. Angiogenesis levels were comparable between Esr-KO and controls. In another PDGFR-β conditional KO mouse (Nestin-KO), PDGFR-β was deleted in neurons and astrocytes from embryonic day 10.5, but was preserved in PC/vSMCs. After MCAO, vascular leakage and infarction volume in Nestin-KO were worse than controls, but partly improved compared with Esr-KO. Astroglial scar formation in both Esr-KO and Nestin-KO was similarly reduced compared with controls after MCAO. These data suggested that PDGFR-β signaling is crucial for neuroprotection, endogenous tissue repair, and functional recovery after stroke by targeting neurons, PC/vSMCs, and astrocytes.

Astrocyte-Derived Tissue Transglutaminase Interacts with Fibronectin: A Role in Astrocyte Adhesion and Migration?

An important neuropathological feature of neuroinflammatory processes that occur during e.g. Multiple Sclerosis (MS) is the formation of an astroglial scar. Astroglial scar formation is facilitated by the interaction between astrocytes and extracellular matrix proteins (ECM) such as fibronectin. Since there is evidence indicating that glial scars strongly inhibit both axon growth and (re)myelination in brain lesions, it is important to understand the factors that contribute to the interaction between astrocytes and ECM proteins. Tissue Transglutaminase (TG2) is a multifunctional enzyme with an ubiquitous tissue distribution, being clearly present within the brain. It has been shown that inflammatory cytokines can enhance TG2 activity. In addition, TG2 can mediate cell adhesion and migration and it binds fibronectin with high affinity. We therefore hypothesized that TG2 is involved in astrocyte-fibronectin interactions. Our studies using primary rat astrocytes show that intracellular and cell surface expression and activity of TG2 is increased after treatment with pro-inflammatory cytokines. Astrocyte-derived TG2 interacts with fibronectin and is involved in astrocyte adhesion onto and migration across fibronectin. TG2 is involved in stimulating focal adhesion formation which is necessary for the interaction of astrocytes with ECM proteins. We conclude that astrocyte-derived TG2 contributes to the interaction between astrocytes and fibronectin. It might thereby regulate ECM remodeling and possibly glial scarring.

Transplantation of Embryonic Neural Stem/Precursor Cells Overexpressing BM88/Cend1 Enhances the Generation of Neuronal Cells in the Injured Mouse Cortex

The intrinsic inability of the central nervous system to efficiently repair traumatic injuries renders transplantation of neural stem/precursor cells (NPCs) a promising approach towards repair of brain lesions. In this study, NPCs derived from embryonic day 14.5 mouse cortex were genetically modified via transduction with a lentiviral vector to overexpress the neuronal lineage-specific regulator BM88/Cend1 that coordinates cell cycle exit and differentiation of neuronal precursors. BM88/Cend1-overexpressing NPCs exhibiting enhanced differentiation into neurons in vitro were transplanted in a mouse model of acute cortical injury and analyzed in comparison with control NPCs. Immunohistochemical analysis revealed that a smaller proportion of BM88/Cend1-overexpressing NPCs, as compared with control NPCs, expressed the neural stem cell marker nestin 1 day after transplantation, while the percentage of nestin-positive cells was significantly reduced thereafter in both types of cells, being almost extinct 1 week post-grafting. Both types of cells did not proliferate up to 4 weeks in vivo, thus minimizing the risk of tumorigenesis. In comparison with control NPCs, Cend1-overexpressing NPCs generated more neurons and less glial cells 1 month after transplantation in the lesioned cortex whereas the majority of graft-derived neurons were identified as GABAergic interneurons. Furthermore, transplantation of Cend1-overexpressing NPCs resulted in a marked reduction of astrogliosis around the lesioned area as compared to grafts of control NPCs. Our results suggest that transplantation of Cend1-overexpressing NPCs exerts beneficial effects on tissue regeneration by enhancing the number of generated neurons and restricting the formation of astroglial scar, in a mouse model of cortical brain injury.

STAT3 is a Critical Regulator of Astrogliosis and Scar Formation after Spinal Cord Injury

Signaling mechanisms that regulate astrocyte reactivity and scar formation after spinal cord injury (SCI) are not well defined. We used the Cre recombinase (Cre)-loxP system under regulation of the mouse glial fibrillary acidic protein (GFAP) promoter to conditionally delete the cytokine and growth factor signal transducer, signal transducer and activator of transcription 3 (STAT3), from astrocytes. After SCI in GFAP-Cre reporter mice, >99% of spinal cord cells that exhibited Cre activity as detected by reporter protein expression were GFAP-expressing astrocytes. Conditional deletion (or knock-out) of STAT3 (STAT3-CKO) from astrocytes in GFAP-Cre-loxP mice was confirmed in vivo and in vitro. In uninjured adult STAT3-CKO mice, astrocytes appeared morphologically similar to those in STAT3+/+ mice except for a partially reduced expression of GFAP. In STAT3+/+ mice, phosphorylated STAT3 (pSTAT3) was not detectable in astrocytes in uninjured spinal cord, and pSTAT3 was markedly upregulated after SCI in astrocytes and other cell types near the injury. Mice with STAT3-CKO from astrocytes exhibited attenuated upregulation of GFAP, failure of astrocyte hypertrophy, and pronounced disruption of astroglial scar formation after SCI. These changes were associated with increased spread of inflammation, increased lesion volume and partially attenuated motor recovery over the first 28 d after SCI. These findings indicate that STAT3 signaling is a critical regulator of certain aspects of reactive astrogliosis and provide additional evidence that scar-forming astrocytes restrict the spread of inflammatory cells after SCI.

Roles of the endogenous VEGF receptors flt-1 and flk-1 in astroglial and vascular remodeling after brain injury

Following trauma to the brain significant changes occur in both the astroglial and vascular components of the neuropil. Angiogenesis is required to re-establish metabolic support and astrocyte activation encompasses several functions including scar formation and the production of growth factors. VEGF has seminal involvement in the process of brain repair and is upregulated during many pathological events. VEGF signaling is regulated mainly through its two primary receptors: flk-1 (KDR/VEGF-R2) is expressed on vascular endothelium and some neurons and flt-1 (VEGF-R1) in the CNS, is expressed predominantly by activated astrocytes. Using an injury model of chronic minipump infusion of neutralizing antibodies (NA) to block VEGF receptor signaling, this study takes advantage of these differences in VEGF receptor distribution in order to understand the role the cytokine plays after brain injury. Infusion of NA to flk-1 caused a significant decrease in vascular proliferation and increased endothelial cell degeneration compared to control IgG infusions but had no effect on astrogliosis. By contrast infusion of NA to flt-1 significantly decreased astroglial mitogenicity and scar formation and caused some increase in endothelial degeneration. Neutralization of the flt-1 receptor function, but not flk-1, caused significant reduction in the astroglial expression of the growth factors, CNTF and FGF by 7days. These data suggest that after CNS injury, endogenous VEGF upregulation (by astrocytes) induces angiogenesis and, by autocrine signaling, increases both astrocyte proliferation and facilitates expression of growth factors. It is likely that VEGF plays an important role in aspects of astroglial scar formation.

Attenuation of astrogliosis by suppressing of microglial proliferation with the cell cycle inhibitor olomoucine in rat spinal cord injury model

Microglial activation/proliferation and reactive astrogliosis are commonly observed and have been considered to be closely relevant pathological processes during spinal cord injury (SCI). However, the molecular mechanisms underlying this microglial–astroglial interaction are still poorly understood. We showed recently that the continuous injection of the cell cycle inhibitor olomoucine not only markedly suppressed microglial proliferation and associated release of pro-inflammatory cytokines, but also attenuated astroglial scar formation and the lesion cavity and mitigated the functional deficits in rat SCI animal model. In this study, we asked whether microglial activation/proliferation plays an initial role and also necessary in maintaining astrogliosis in SCI model. Our results showed that traumatic induced microglial activation/proliferation precedes astrogliosis, and the up-regulated GFAP expression at both mRNA and protein levels was temporally posterior to the microglial activation. Furthermore, when the cell cycle inhibitor olomoucine was administered only once 1 h post-SCI that should selectively suppress microglial proliferation, the subsequent SCI induced increase in GFAP expression at 1, 2 and 4 weeks was significantly attenuated, suggesting that microglial activation/proliferation played an important role for the later onset astrogliosis after SCI. Consistent with the results that microglial proliferation always precedes astroglial proliferation and there is at present no evidence of other astroglial precursors, which as always does not mean that they will not be uncovered by further searching, and in view of the fact that microglial-derived pro-inflammatory cytokines promote astrogliosis as we reported recently, these findings together suggest that by release of cytokines and other soluble products, the early onset microglial activation/proliferation can significantly influence the subsequent development of reactive astrogliosis and glial scar formation in SCI animal model.

Cell cycle inhibition attenuates microglia induced inflammatory response and alleviates neuronal cell death after spinal cord injury in rats

The spinal cord is well known to undergo inflammatory reactions in response to traumatic injury. Activation and proliferation of microglial cells, with associated proinflammatory cytokines expression, plays an important role in the secondary damage following spinal cord injury. It is likely that microglial cells are at the center of injury cascade and are targets for treatments of CNS traumatic diseases. Recently, we have demonstrated that the cell cycle inhibitor olomoucine attenuates astroglial proliferation and glial scar formation, decreases lesion cavity and mitigates functional deficits after spinal cord injury (SCI) in rats [Tian, D.S., Yu, Z.Y., Xie, M.J., Bu, B.T., Witte, O.W., Wang, W., 2006. Suppression of astroglial scar formation and enhanced axonal regeneration associated with functional recovery in a spinal cord injury rat model by the cell cycle inhibitor olomoucine. J. Neurosci. Res. 84, 1053–1063]. Whether neuroprotective effects of cell cycle inhibition are involved in attenuation of microglial induced inflammation awaits to be elucidated. In the present study, we sought to determine the influence of olomoucine on microglial proliferation with associated inflammatory response after spinal cord injury. Tissue edema formation, microglial response and neuronal cell death were quantified in rats subjected to spinal cord hemisection. Microglial proliferation and neuronal apoptosis were observed by immunofluorescence. Level of the proinflammatory cytokine interleukin-1β (IL-1β) expression in the injured cord was determined by Western blot analysis. Our results showed that the cell cycle inhibitor olomoucine, administered at 1 h post injury, significantly suppressed microglial proliferation and produced a remarkable reduction of tissue edema formation. In the olomoucine-treated group, a significant reduction of activated and/or proliferated microglial induced IL-1β expression was observed 24 h after SCI. Moreover, olomoucine evidently attenuated the number of apoptotic neurons after SCI. Our findings suggest that modulation of microglial proliferation with associated proinflammatory cytokine expression may be a mechanism of cell cycle inhibition-mediated neuroprotections in the CNS trauma.

Extracellular regulators of axonal growth in the adult central nervous system.

Robust axonal growth is required during development to establish neuronal connectivity. However, stable fibre patterns are necessary to maintain adult mammalian central nervous system (CNS) function. After adult CNS injury, factors that maintain axonal stability limit the recovery of function. Extracellular molecules play an important role in preserving the stability of the adult CNS axons and in restricting recovery from pathological damage. Adult axonal growth inhibitors include a group of proteins on the oligodendrocyte, Nogo-A, myelin-associated glycoprotein, oligodendrocyte-myelin glycoprotein and ephrin-B3, which interact with axonal receptors, such as NgR1 and EphA4. Extracellular proteoglycans containing chondroitin sulphates also inhibit axonal sprouting in the adult CNS, particularly at the sites of astroglial scar formation. Therapeutic perturbations of these extracellular axonal growth inhibitors and their receptors or signalling mechanisms provide a degree of axonal sprouting and regeneration in the adult CNS. After CNS injury, such interventions support a partial return of neurological function.

Suppression of astroglial scar formation and enhanced axonal regeneration associated with functional recovery in a spinal cord injury rat model by the cell cycle inhibitor olomoucine

It is well established that axons of the adult mammalian CNS are capable of regrowing only a limited amount after injury. Astrocytes are believed to play a crucial role in the failure to regenerate, producing multiple inhibitory proteoglycans, such as chondroitin sulphate proteoglycans (CSPGs). After spinal cord injury (SCI), astrocytes become hypertrophic and proliferative and form a dense network of astroglial processes at the site of lesion constituting a physical and biochemical barrier. Down-regulations of astroglial proliferation and inhibitory CSPG production might facilitate axonal regeneration. Recent reports indicated that aberrant activation of cell cycle machinery contributed to overproliferation and apoptosis of cells in various insults. In the present study, we sought to determine whether a cell cycle inhibitior, olomoucine, would decrease neuronal cell death, limit astroglial proliferation and production of inhibitory CSPGs, and eventually enhance the functional compensation after SCI in rats. Our results showed that up-regulations of cell cycle components were closely associated with neuronal cell death and astroglial proliferation as well as the production of CSPGs after SCI. Meanwhile, administration of olomoucine, a selective cell cycle kinase (CDK) inhibitor, has remarkably reduced the up-regulated cell cycle proteins and then decreased neuronal cell death, astroglial proliferation, and accumulation of CSPGs. More importantly, the treatment with olomoucine has also increased expression of growth-associated proteins-43, reduced cavity formation, and improved functional deficits. We consider that suppressing astroglial cell cycle in acute SCIs is beneficial to axonal growth. In the future, therapeutic strategies can be designed to achieve efficient axonal regeneration and functional compensation after traumatic CNS injury.

Cell cycle inhibition provides neuroprotection and reduces glial proliferation and scar formation after traumatic brain injury.

Traumatic brain injury (TBI) causes neuronal apoptosis, inflammation, and reactive astrogliosis, which contribute to secondary tissue loss, impaired regeneration, and associated functional disabilities. Here, we show that up-regulation of cell cycle components is associated with caspase-mediated neuronal apoptosis and glial proliferation after TBI in rats. In primary neuronal and astrocyte cultures, cell cycle inhibition (including the cyclin-dependent kinase inhibitors flavopiridol, roscovitine, and olomoucine) reduced up-regulation of cell cycle proteins, limited neuronal cell death after etoposide-induced DNA damage, and attenuated astrocyte proliferation. After TBI in rats, flavopiridol reduced cyclin D1 expression in neurons and glia in ipsilateral cortex and hippocampus. Treatment also decreased neuronal cell death and lesion volume, reduced astroglial scar formation and microglial activation, and improved motor and cognitive recovery. The ability of cell cycle inhibition to decrease both neuronal cell death and reactive gliosis after experimental TBI suggests that this treatment approach may be useful clinically.

Decorin suppresses neurocan, brevican, phosphacan and NG2 expression and promotes axon growth across adult rat spinal cord injuries.

The formation of misaligned scar tissue by a variety of cell types expressing multiple axon growth inhibitory proteoglycans presents a physical and molecular barrier to axon regeneration after adult spinal cord injuries. Decorin is a small, leucine-rich proteoglycan that has previously been shown to reduce astrogliosis and basal lamina formation in acute cerebral cortex stab injuries. We have therefore tested whether mini pump infusion of hr-decorin into acute stab injuries of the adult rat spinal cord can not only inhibit formation of an astroglial limitans but also deposition of the axon growth inhibitory proteoglycans neurocan, NG2, phosphacan and brevican. Combined immunohistochemical and quantitative Western blot analysis revealed major reductions in levels of core protein expression (>80% for 130-kDa neurocan, 145/80-kDa brevican, 300-kDa phosphacan) and immunoreactivity for all four chondroitin sulfate proteoglycans (CSPGs) within decorin-treated injuries compared with untreated controls. Astrogliosis within lesion margins and the accumulation of OX42+ macrophages/microglia within lesion centres were also significantly reduced. These decorin-induced changes in scar formation combined to promote the striking ability of axons from microtransplanted adult sensory neurons to enter, grow within and exit decorin-infused spinal cord injuries, in sharp contrast to the complete failure of axons to cross untreated, CSPG-rich lesions. Decorin pretreatment of meningial fibroblasts in vitro also resulted in a three-fold increase in neurite outgrowth from co-cultured adult sensory neurons and suppression of NG2 immunoreactivity. The ability of decorin to promote axon growth across acute spinal cord injuries via a coordinated suppression of inflammation, CSPG expression and astroglial scar formation make decorin treatment a promising component of future spinal cord regeneration strategies.

Dynamic changes of magnetic resonance imaging abnormalities in relation to inflammation and glial responses after photothrombotic cerebral infarction in the rat brain.

This investigation analyzed the potential of high-resolution magnetic resonance imaging (MRI) at a field strength of 7T to depict leukocyte infiltration and glial responses after focal cerebral ischemia induced by photothrombotic occlusion of cerebral microvessels. For this purpose we superimposed multiparametric MRI (apparent diffusion coefficient, T2, perfusion-weighted, and gadolinium-DTPA-enhanced T1-weighted imaging) on tissue sections stained for phagocytes and astrocytes and, moreover, assessed the regional distribution of tissue pH and ATP content by invasive biochemical methods. Comparing the histological data with the various MRI parameters, high-resolution MRI did not allow a spatial discrimination between distinct areas of phagocyte accumulation or astroglial scar formation, based on image contrast or even quantitative parameter value differences. However, MRI parameters underwent characteristic changes and differentiated distinct stages of tissue remodeling between days 3 and 14 after photothrombosis. Low apparent diffusion coefficient (ADC) and high T2 values indicated an early stage (3 days) with necrosis and beginning glial activation. Normal ADC and reduced T2 elevation characterized an infarct with advanced glial activation and infiltration of hematogenous cells at 7 days after photothrombosis. Heterogeneous ADC together with T2 elevation reflected a late infarct stage (14 days) when pseudocystic degeneration and scar formation had occurred.

Chondroitin sulfates expressed on oligodendrocyte-derived tenascin-R are involved in neural cell recognition. Functional implications during CNS development and regeneration.

Tenascin-R (TN-R), an extracellular matrix constituent of the central nervous system (CNS), has been implicated in a variety of cell-matrix interactions underlying axon growth inhibition/guidance, myelination and neural cell migration during development and regeneration. Although most of the functional analyses have concentrated exclusively on the role of the core protein, the contribution of TN-R glycoconjugates present on many potential sites for N- and O-glycosylation is presently unknown. Here we provide first evidence that TN-R derived from whole rat brain or cultured oligodendrocytes expresses chondroitin sulfate (CS) glycosaminoglycans (GAGs), i.e., C-4S and C-6S, that are recognized by CS-56, a CS/dermatan sulfate-specific monoclonal antibody. Based on different in vitro approaches utilizing substrate-bound glycoprotein, we found that TN-R-linked CS GAGs (1) promote oligodendrocyte migration from white matter microexplants and increase the motility of oligodendrocyte lineage cells; (2) similar to soluble CS GAGs, induce the formation of glial scar-like structures by cultured cerebral astrocytes; and (3) contribute to the antiadhesive properties of TN-R for neuronal cell adhesion in an F3/F11-independent manner, but not to neurite outgrowth inhibition, by mechanism(s) sensitive to chondroitinase or CS-56 treatments. Furthermore, after transection of the postcommissural fornix in adult rat, CS-bearing TN-R was found to be stably upregulated at the lesion site. Our findings suggest the functional impact of TN-R-linked CS on neural cell adhesion and migration during brain morphogenesis and the contribution of TN-R to astroglial scar formation (CS-dependent) and axon growth inhibition (CS-independent), i.e., suppression of axon regeneration after CNS injury.

Reactive microglia in cerebral ischaemia: an early mediator of tissue damage?

Microglial cell activation is a rapidly occurring cellular response to cerebral ischaemia. Microglia proliferate, are recruited to the site of lesion, upregulate the expression of several surface molecules including major histocompatibility complex class I and II antigens, complement receptor and the amyloid precursor protein (APP) as well as newly expressed cytokines, e.g. interleukin-1 and transforming growth factor beta 1. The ischaemia-induced production of APP may contribute to amyloid deposition in the aged brain under conditions of hypofusion. Ultrastructurally, microglia transform into phagocytes removing necrotic neurons but still respecting the integrity of eventually surviving neurons even in the close vicinity of necrotic neurons. Microglial activation starts within a few minutes after ischaemia and thus precedes the morphologically detectable neuronal damage. It additionally involves a transient generalized response within the first 24 hours post-ischaemia even at sites without eventual neuronal cell death. In functional terms, the microglial reaction appears to be a double-edged sword in ischaemia. Activated microglia may exert a cytotoxic effector function by releasing reactive oxygen species, nitric oxide, proteinases or inflammatory cytokines. All of these cytotoxic compounds may cause bystander damage following ischaemia. Pharmacological suppression of microglial activation after ischaemia has accordingly attenuated the extent of cell death and tissue damage. However, activated microglia support tissue repair by secreting factors such as transforming growth factor beta 1 which may limit tissue damage as well as suppress astroglial scar formation. In line with ultrastructural observations microglial activation in ischaemia is a strictly controlled event. By secreting cytokines and growth factors activated microglia most likely serve seemingly opposed functions in ischaemia, i.e. maintenance as well as removal of injured neurons. Post-ischaemic pharmacological modulation of microglial intervention in the cascade of events that lead to neuronal necrosis may help to improve the structural and functional outcome following CNS ischaemia.

Changes in astroglial scar formation in rat optic nerve as a function of development

Although astroglial scar formation is a common response to almost any type of injury to the adult central nervous system, lesions in fetal and neonatal rats have been reported to induce little or no scar formation. To examine this developmental difference further, rats ranging in age from 1 to 65 days postnatal were unilaterally enucleated, a surgical procedure that causes the axons in the optic nerve to degenerate. The optic nerves were processed for light and electron microscopy at times ranging from 7 to 365 days postenucleation. Pronounced and permanent glial scars were formed in every age group examined, including the neonates. However, the time course for removal of the degenerating axonal debris and formation of a compact, debris-free glial scar varied as a function of developmental age. In neonatal rats, a compact glial scar formed in 1-2 weeks whereas 3-5 months were required for compact glial scar formation in juveniles and adults. Changes in cross-sectional area were also associated with optic nerve degeneration and glial scar formation. Whereas lesioned neonatal optic nerves underwent little change in area, there was a substantial decrease in area in the juvenile and adult. Morphometric analysis showed that irrespective of the age of the animal at the time of enucleation, the final area of the compact glial scar was 10-20% of the unlesioned adult control. These results suggest that conflict in the literature over the ability of neonatal astrocytes to form a glial scar may be due to the nature of the lesion or the method of detection since astrocytes in the neonatal rat optic nerve clearly have the capacity to become reactive and form a glial scar.

Kainic acid induced seizures: Neurochemical and histopathological changes

Behavioural, histopathological and neurochemical changes induced by systemic injection of kainic acid (10mg/kg, s.c.) were investigated in rats. The most pronounced behavioural changes were strong immobility (“catatonia”), increased incidence of “wet dog shakes”, and long-lasting generalized tonic-clonic convulsions. The behavioural symptoms were fast in their onset and lasted for several hours. Two distinct phases of histopathological and neurochemical changes were observed. (1) Early partially reversible changes were seen up to 3 h after kainic acid injection. They consisted of shrinkage and pycnosis of neuronal perikarya together with swelling of dendrites and axon terminals. These changes were accompanied by generalized signs of edema throughout the whole brain. Neurochemically there was a marked decrease in noradrenaline levels (up to 70%) and an increase in levels of 5-hydroxyindoleacetic acid, 3,4-dihydroxyphenylacetic acid and homovanillic acid (up to 200%) in all analysed brain regions, suggesting a strongly increased firing rate of aminergic neurones during the period of generalized seizures. These histological and neurochemical changes were found in all the brain regions examined; they were greatly reduced or only sporadically seen after 1–3 days, when the animals had recovered from the seizures. (2) Late irreversible changes developed 24 h and later following kainic acid injection. They consisted of incomplete tissue necrosis with loss of nerve cells and oligodendrocytes, demyelination, astroglial scar formation, small perivenous hemorrhages and extensive vascular sprouting. The changes were restricted to the pyriform cortex, amygdala, hippocampus (most pronounced in the CA1 sector), gyrus olfactorius lateralis, bulbus olfactorius and tuberculum olfactorium. Neurochemically, a selective decrease was seen in choline acetyltransferase activity (40%) of the amygdala/pyriform cortex area, and of glutamate decar☐ylase activity in the dorsal hippocampus (45%) and amygdala/pyriform cortex (55%). No such changes were found in the frontal cortex and the striatum/pallidum. Since at these later time periods the widespread early changes in monoamine metabolism were mostly normalized, loss of acetylcholine and γ-aminobutyric acid neurons in the affected brain regions represented a selective neurochemical change typical for this stage of kainic acid action. The observed neurochemical and histopathological changes may be directly related to tne excitotoxic and convulsive properties of kainic acid. However, brain edema resulting in herniation damage of the basal portions of the brain in addition to disturbances of microcirculation and anoxic-ischemic brain damage appear to be additional factors important in the pathogenesis of the late irreversible changes.