Glial response in the central nervous system of cats with feline infectious peritonitis

Abstract

Objectives

The aim of the study was to evaluate central nervous system (CNS) lesions in non-effusive and effusive cases of feline infectious peritonitis (FIP) regarding aspects related to astrocytic and microglial reactions.

Methods

Five necropsied cats that were naturally infected with FIP virus, confirmed by reverse transcriptase polymerase chain reaction and immunohistochemistry, with different intensities of CNS lesions, were studied. Brain and cerebellum were evaluated by light microscopy and immunohistochemistry for glial fibrillary acidic protein (GFAP) and vimentin to assess astrocytic morphology, and lectin histochemistry for Ricinus communis agglutinin-I (RCA-I) to detect microglia was performed to evaluate the glial response in the CNS of cats with FIP.

Results

An important astrocytic response in many areas of the CNS of all cats, including the periventricular areas of lateral ventricles and fourth ventricle, the molecular layer of the cerebellum and cerebral cortex, was visualized. This astrocytic reactivity was associated with areas of granulomatous or pyogranulomatous vasculitis/perivasculitis in most cases, and it was characterized by multifocal to coalescing astrocytosis and astrogliosis with an increase in the expression of intermediate filaments, such as GFAP. However, astrocytes exhibited strong vimentin expression in neuroparenchyma with severe inflammatory and necrotic changes, but GFAP expression was mild or absent in these cases. A microglial response was present only in severe lesions, and RCA-I expression was detected primarily in gitter cells and resting microglia.

Conclusions and relevance

The present study indicates a strong astrocytic response, including the presence of many less differentiated vimentin-positive astrocytes and gitter cells positive for RCA-1 in severe lesions in the CNS of cats with FIP.

Introduction

Feline infectious peritonitis (FIP) is a widely distributed and well-known disease caused by a feline coronavirus (FCoV), which is one of the primary infectious causes of death in cats. ¹ FIP lesions are characterized by a fibrinous or granulomatous serositis that is associated with effusions in body cavities and/or pyogranulomatous lesions in many organs, including the central nervous system (CNS). ² Lesions in the CNS occur in the leptomeninges of the brain and spinal cord, and they are characterized by granulomatous to pyogranulomatous meningitis, ependymitis or periventriculitis and choroiditis of varying degrees of severity. ³ Degenerative and necrotic lesions in the CNS parenchyma can occur, and they are likely related to vasculitis. ⁴

The first report of parenchymal lesions in the CNS in cats with FIP included scattered glial nodules in the brain. ⁵ A complete obliteration of the ependyma by inflammatory cells with reactive astrocytosis and inflammatory infiltrates within the neuroparenchyma subjacent to leptomeninges can also occur.^3,6 Many studies have characterized the inflammatory component in the CNS of cats with FIP,^7–9 but few have attempted to evaluate the necrotic and degenerative changes in the neuroparenchyma of cats with FIP regarding glial reactivity. Therefore, this study evaluated CNS lesions in non-effusive and effusive cases of FIP by evaluating aspects related to astrocytic and microglial reactions.

Materials and methods

Animals and tissues

The study was performed on five cats with neurological lesions that were routinely necropsied and naturally infected by the feline infectious peritonitis virus (FIPV). The diagnosis of FIP was based mainly on histological lesions, by the presence of FCoV mRNA of the membrane (M) gene in the CNS by reverse transcriptase polymerase chain reaction (RT-PCR) ¹⁰ and detection of viral antigens by immunohistochemistry (IHC).

All cats underwent a necropsy, and samples of several organs and the CNS (except the spinal cord) were collected and fixed in 10% buffered formalin and embedded in paraffin. Brain sections were taken from the telencephalon at the level of frontal cortex and piriform lobe (hippocampus); mesencephalon at the level of rostral and caudal colliculus; metencephalon at the level of cerebellar peduncles (pons); and from medulla oblongata (myelencephalon). Sections (5 µm) were stained using hematoxylin and eosin. Histological lesions were classified as mild, moderate or severe.

Samples of the CNS (brain and cerebellum) were also flash-frozen in liquid nitrogen and stored at −80ºC. RNA extraction and RT-PCR were performed as previously described. ¹⁰

IHC and lectin histochemistry

Sections (5 µm) were submitted to IHC and lectin histochemistry using the streptavidin–biotin–peroxidase method (LSAB; Dako). The sections were dewaxed, rehydrated through graded alcohols and submitted to endogenous peroxidase inactivation by methanol with hydrogen peroxide (3%). Antigen retrieval was performed in a water bath at 98ºC in citrate buffer (pH 6.0) for 20 mins. The following antibodies were used to detect the following antigens: glial fibrillary acidic protein (GFAP) and vimentin for astrocytes. Biotinylated lectin Ricinus communis agglutinin-I (RCA-I) was used to detect microglia. The sections were incubated in a humid chamber at 4ºC for 16–18 h with the following primary antibodies: anti-GFAP (rabbit polyclonal, 1:400; Dako), anti-vimentin (mouse monoclonal, clone V9, 1:100; Dako) and RCA-I (5 µg/ml; Vector Laboratories). Subsequently, the slides were incubated with biotinylated secondary antibodies (LSAB; Dako) followed by streptavidin–peroxidase (LSAB; Dako), both for 30 mins. In sections incubated with biotinylated RCA-I, only the streptavidin–peroxidase solution (LSAB; Dako) was used for 30 mins. The binding between antigens and antibodies was visualized using the chromagen Vector Red (Vector NovaRed; Vector Laboratories) according to manufacturer’s instructions, followed by slight counterstain with hematoxylin. Primary antibodies were substituted by homologous non-immune sera as negative controls. Sections of cat brain and cerebellum without histological alterations were used as controls.

Additionally, immunohistochemical staining for FIPV was performed on sections of brain and cerebellum using a monoclonal antibody (Anti-FIPV3-70 antibody; Custom Monoclonals International) as previously described. ⁶

Anti-GFAP and anti-vimentin antibodies were used for astrocytic evaluations, and the following morphological aspects of the cell were considered: size and shape of cell bodies, size of nucleus, distribution of chromatin, and the number and length of processes per cell. The intensity of the IHC reaction was compared between GFAP and vimentin labelling. The pattern of immunoreactivity was compared between the cats with FIP, which had different intensities of CNS lesions.

Results

Animals and gross pathology

The age and sex of the cats are listed in Table 1. All animals were intact domestic shorthair cats. Veterinary medical records were available in three cases (cats 1, 2 and 3). The clinical course of disease varied from 2–6 weeks and all three cats presented with progressive weight loss, prostration, inappetence and CNS signs. Neurological signs were most severe in cat 1 and were characterized by hyperesthesia, blindness, head tilt, circling and ataxia that progressed to lateral recumbence. Cat 2 presented with ataxia and cat 3 had posterior paralysis of 6 weeks’ duration.

Table 1.

Clinical signs, distribution of lesions in the central nervous system (CNS) and other organs affected in cats naturally infected by feline infectious peritonitis virus

Cat	Age (months)	Sex	Form of disease	Neurological signs	Localization of CNS lesions	Other affected organs*
1	3	F	Non-effusive	Hyperesthesia, blindness, head tilt, circling, ataxia and lateral recumbency	Leptomeninges, periventricular areas of lateral ventricles and fourth ventricle, hippocampus, molecular layer and white matter of cerebellum, and cerebral cortex	Lung, heart, liver and kidneys
2	12	F	Effusive	Ataxia	Leptomeninges and periventricular areas of lateral and fourth ventricles	Lung, heart, pleural surfaces, kidneys, mesenteric lymph nodes, and serosa of large and small intestine
3	6	M	Non-effusive	Ataxia and hindlimb paralysis	Leptomeninges, cerebral cortex, molecular, Purkinje and granular layer of cerebellum, and periventricular areas of lateral and fourth ventricles	Liver and kidney
4	4	M	Effusive	NR	Leptomeninges	Parietal pleura and serosa of lungs and epicardium
5	7	F	Non-effusive	NR	Leptomeninges and periventricular areas of fourth ventricle	None

^*Other organs were considered affected when vasculitis/perivasculitis or pyogranulomatous/granulomatous lesions were present

M = male; F = female; NR = not recorded

The mRNA of the FIVP M gene was detected in samples of brain and cerebellum, except for cat 3, in which FIVP mRNA was found only in cerebellum.

Macroscopic lesions in the CNS were characterized by opacification of meninges (cat 1) and leptomeningeal vessel congestion (cats 1, 2 and 3).

Histology

All cats in this study exhibited inflammatory lesions with varying degrees of severity. Degenerative/necrotic processes of neuroparenchyma, visualized in four cats, were always associated with inflammatory changes. The distribution of histological lesions in the CNS is listed in Table 1.

The histological lesions were characterized as a granulomatous or pyogranulomatous vasculitis and/or perivasculitis. Inflammatory infiltrates were composed of a large number of macrophages in all cases. The amount of neutrophils, lymphocytes and plasma cells varied among cases. Perivascular spaces, mainly around venules, were expanded by large amounts of inflammatory cells in cases with severe vasculitis, and the walls of these vessels were disrupted by the inflammatory infiltrate, fibrin and cellular debris. Endothelial cells were hypertrophied (reactive), and some venules presented a narrowing of the lumen with occasional thrombus formation. The perivasculitis/vasculitis was located mainly in leptomeninges (Table 1), which occasionally extended to the adjacent neuroparenchyma of the cerebral cortex (cats 1 and 3), the molecular layer of cerebellum (cat 1), and the molecular, Purkinje and granular layers of the cerebellum of cat 3. Other affected areas included the white matter of periventricular areas of lateral ventricles of cats 1, 2 and 3, extension to the hippocampus (cat 1) and white matter adjacent to the fourth ventricle and extending to the white matter of the cerebellum (cats 1, 2, 3 and 5).

There was a severe, diffuse spongiosis with liquefied areas (liquefactive necrosis) that was associated with a large amount of reactive astrocytes in the white matter of periventricular areas of lateral ventricles in cat 1. There was also complete effacement of the ependyma by macrophages, neutrophils and some reactive astrocytes that resembled gemistocytes. There was a moderate number of foam macrophages (gitter cells) in areas of liquefactive necrosis. Neurons of the hippocampus near areas of vasculitis in this cat were red, angular and included condensed nuclei (neuronal necrosis) (Figure 1). Additionally, the white matter of cerebellum adjacent to the fourth ventricle in cats 1, 2, 3 and 5 exhibited locally extensive areas similar to the periventricular areas of cat 1, and these areas were also associated with multifocal vacuolization of the white matter. Degenerative and inflammatory lesions were also present in adjacent areas of lateral ventricles in the cats 2 and 3, but they were mild compared with cat 1. Perivascular spaces were wide as a result of fluid leakage (edema), and it was mild in cats 2 and 3, moderate in cat 2 and severe in cat 1.

Figure 1.

Cat 1. Neuronal necrosis with pyogranulomatous vasculitis in the hippocampus. Hematoxylin and eosin staining (bar = 50 µm)

IHC and lectin histochemistry

In the brain and cerebellum of all cats, FIPV antigen was detected in the cytoplasm of mononuclear cells morphologically consistent with macrophages within the leptomeninges (Figure 2). A small number of similar cells located in Virchow Robin space of vessels in periventricular areas were also positive for FIP antigen.

Figure 2.

Cat 4. Macrophages within the leptomeninges exhibit strong immunolabeling for feline infectious peritonitis virus antigen. Immunohistochemistry (bar = 100 µm)

Astrocytic reactions were evaluated using GFAP and vimentin immunolabeling, which was visualized in different areas of the CNS in all cats of the present study. Astrocytosis and astrogliosis were mainly detected in the periventricular areas of the lateral ventricles (cats 1, 2 and 3) and fourth ventricle (cats 1, 2, 3 and 5), the granular layer of cerebellum (cat 3), and the molecular layer of cerebellum and cerebral cortex of all cats.

The gray matter was primarily associated with leptomeningitis and perivasculitis/vasculitis of the neuroparenchyma in the cerebral cortex, where there was an increase in the number of protoplasmic astrocytes (astrocytosis). This astrocytosis was moderate in cats 1, 2 and 3, and mild in the other cats. Astrocytes in these areas had larger nuclei with finely granular chromatin and prominent nucleoli, with an abundant cytoplasm that was strongly and well delimited by GFAP immunolabeling (astrogliosis). Rare reactive astrocytes were binucleated. Astrocytic processes were increased in number, shorter and exhibited a larger diameter than other areas of cerebral cortex in which histological lesions were not present. Strong immunolabeling for vimentin was present in protoplasmic astrocytes of the cerebral cortex only in cat 1. Astrocytosis and astrogliosis were accompanied by strong GFAP immunolabeling of the glia limitans.

Astrocytosis and astrogliosis were more severe in periventricular areas than in areas in the cerebral cortex. The astrocytic reaction in white matter adjacent to the lateral ventricles was characterized by the presence of large numbers of plump astrocytes with abundant cytoplasm and large vesicular nuclei. These alterations were most severe in cat 1. GFAP immunolabeling in the cytoplasm of these astrocytes was mild or absent, and astrocytic processes involving blood vessels with vasculitis presented mild GFAP immunolabeling (Figure 3). GFAP immunolabeling in these areas was strong only in the cytoplasm of gemistocytes, which were located near the absent ependyma. Vimentin immunolabeling was strong in a large number of astrocytes in periventricular areas (Figure 4). This immunolabeling was homogeneous, and it appeared in the entire cytoplasm, including the astrocytic processes. Therefore, vimentin expression in these areas was stronger than GFAP. Immunolabeling for GFAP and vimentin in periventricular areas of the fourth ventricle (cats 1, 2, 3 and 5) were similar to the lateral ventricles. However, there were more gemistocytes that were positive for both GFAP and vimentin in some cats (cats 2, 3 and 5). Additionally, there was strong vimentin immunolabeling in astrocytes in the hippocampus in cat 1.

Figure 3.

Cat 1. In white matter of the periventricular area of the lateral ventricle, immunolabeling for GFAP in the cytoplasm and processes of plump astrocytes is mild or absent. Immunohistochemistry (bar = 50 µm)

Figure 4.

Cat 1. Strong vimentin expression in the cytoplasm of plump astrocytes of periventricular area of lateral ventricle. Immunohistochemistry (bar = 50 µm)

Moderate GFAP immunolabeling was present in Bergmann glia near areas of leptomeningitis in the cerebellum of cats 1, 2 and 3 (Figure 5). The processes of Bergmann glia were thicker and extended perpendicularly through the entire molecular layer. In contrast, vimentin immunolabeling in these astrocytes was stronger (Figure 6). Bergmann glia processes exhibited mild or absent immunolabeling for GFAP and vimentin in areas where leptomeningitis was not present. Vimentin immunolabeling in Bergmann glia was mild in cats 4 and 5. A large number of astrocytes in the cerebellum folia white matter (cat 1) exhibited strong vimentin immunolabeling, but astrocytes with this characteristic were rarely found in the white matter of cerebellum folia in cat 2.

Figure 5.

Cat 1. Moderate glial fibrillary acidic protein expression in Bergmann glia adjacent to areas of leptomeningitis. Immunohistochemistry (bar = 100 µm)

Figure 6.

Cat 1. Strong vimentin expression in Bergmann glia and also in mononuclear cells within the leptomeninges. Immunohistochemistry (bar = 100 µm)

Astrocytes positive for vimentin were present in the brainstem at the level of cerebellar peduncles, rostral and caudal colliculus of cat 3 and near the glia limitans of the pons and medulla oblongata (cats 1 and 3). Inflammatory changes were not present in these brainstem areas where positive astrocytes for vimentin were found.

RCA-I lectin labeling was detected in resident microglia in the resting state of all cats, and these cells were characterized by thin and weakly stained processes. There was strong labeling for RCA-I lectin in gitter cells in the areas of periventriculitis in cat 1 (Figure 7). There was mild microgliosis in these areas in other cats with periventriculitis with few microglia stained for RCA-I.

Figure 7.

Cat 1. Gitter cells in periventricular area of lateral ventricle are strongly positive for RCA-I lectin. Immunohistochemistry (bar = 50 µm)

Discussion

This study describes the features of lesions in the CNS parenchyma and the glial response in cats with FIP. Inflammatory lesions in the CNS lesions in cats of the present study were similar as previously described for neurological FIP.^5–9,11 Leptomeningitis is generally restricted to the meninges, with only modest extension to the cortex, but marked encephalitis with necrosis was usually present in subependymal areas. ⁵ In contrast, some cats of the present study exhibited an important involvement of the cerebral cortex and hippocampus (cat 1), and molecular, Purkinje and granular layers of the cerebellum of cat 3. Glial nodules were described previously in brain parenchyma of cats with neurological FIP. ⁵ However, this type of lesion was not found in the present study.

Regarding the glial response, the results of the present study revealed a prominent astrocytic response in CNS parenchyma with degenerative and necrotic lesions and also in areas without parenchymal lesions. Astrocytic reactivity was present in all cats in different areas of the CNS, including the cerebral cortex, ventricular and periventricular areas, cerebellum and brainstem. The astrocytic response was characterized by an increase in the number of astrocytes (astrocytosis), an increased expression of intermediate filaments (GFAP and/or vimentin) and an increase in the length, complexity and branching of astrocytic processes (astrogliosis). This astrocytic reactivity was present primarily in areas with degenerative and necrotic changes in neuroparenchyma and in areas adjacent to leptomeningitis and/or perivasculitis/vasculitis. However, reactive astrocytes positive for vimentin were detected in areas without histological lesions. Astrocytic activation using GFAP expression was described previously in many domestic animal disorders that include inflammatory and degenerative changes of the CNS, such as in dogs with distemper,^12–14 and leishmaniosis, ¹⁵ cattle with rabies ¹⁶ and horses with leukoencephalomalacia. ¹⁷ Complete replacement of ependymal cells by histiocytic and lymphocytic infiltrates with periventricular reactive astrocytosis, ³ and scattered glial nodules, ⁵ can occur in severe cases of neurological FIP. Astrogliosis with an increase of GFAP expression is also described in the gray matter of cortical structures of the CNS of cats infected with feline immunodeficiency virus. ¹⁸

The present study demonstrated diminished GFAP expression in a large number of plump astrocytes in periventricular areas with severe degenerative and necrotic changes in neuroparenchyma and areas with a prominent astrocytic response. In contrast, these astrocytes were strongly immunolabeled for vimentin. Astrocytes were positive for vimentin and GFAP in an area that extended from subependymal areas of the lateral ventricles to the hippocampus and cerebral cortex, where neuroparenchyma lesions were less severe. Diminished GFAP expression with a concomitant increase in vimentin expression such as was seen in our study has also been observed in other diseases with severe parenchymal lesions; ie, old dog encephalitis. ¹⁹ Vimentin expression with or without concomitant GFAP expression reflects a reactive or degenerative change in astrocytes. ¹² Vimentin expression in astrocytes was reported previously in CNS areas with lesions, but it was absent in normal CNS.^12,20,21 However, astrocytes in the corpus callosum, hippocampus and Bergmann glia express vimentin in normal conditions. ²² In the present study, vimentin expression in astrocyte cytoplasm was mainly detected in areas with inflammatory and/or degenerative changes. Vimentin is mainly expressed in maturing astrocytes, and its expression in adult astrocytes may be interpreted as a transitory shift to an immature phenotype, which might represent a reactive change. ²³ Diminished GFAP expression with an increase in the co-expression of vimentin and GFAP or vimentin alone is also described in canine distemper demyelinating leukoencephalitis (DL). ²¹ Vimentin-immunoreactive astrocytes may also be interpreted as a recruitment of immature astrocytes to sites of advanced DL lesions. ²¹ Cats of the present study exhibited increased vimentin expression in astrocytes that was associated with a diminished or absent expression of GFAP, primarily in sites of severe lesions of neuroparenchyma, which is as similar to canine distemper.

Vimentin-positive astrocytes were also detected in areas of the brainstem where there were no inflammatory or degenerative changes in the present study. The proximity of severe periventricular lesions in the fourth ventricle and leptomeningitis could explain the astrocytic reactivity in the brainstem, although there were no important lesions in the same histological section. Similarly, reactive astrocytes were present in extralesional sites in horses with leukoencephalomalacia, ¹⁷ and cattle with rabies. ¹⁶ Astrocytes in the corpus callosum and hippocampus and Bergmann glia may also co-express vimentin and GFAP. ²² However, in the present study, a severe astrocytic reaction was observed in Bergmann glia that was associated with strong and moderate expression of vimentin and GFAP, respectively. The astrocytic response in this case occurred mainly in areas of leptomeningitis. Vimentin and GFAP expression was absent in areas where leptomeningitis was absent, and mild expression was rarely observed in Bergmann glia in areas without inflammatory changes. Vimentin expression in astrocytes of the hippocampus in cats with FIP was clearly associated with neuroparenchymal lesions, astrocytosis and astrogliosis.

Reactive astrogliosis has been described in many pathological conditions of the CNS, including neurodegenerative, inflammatory and demyelinating disorders and viral infections. ²⁴ The role of astrocytes in FIP pathogenesis is not known. Some cytokines, such as IL-6 and IL-1β, and chemokines, such as MIP-1α and RANTES, are expressed in the CNS of cats with FIP. ¹¹ MIP-1α and RANTES, which are chemoattractants for macrophages and lymphocytes, are also produced by intrinsic cells of the CNS, including astrocytes. ²⁵ Cats with FIP vasculitis exhibit intravascular monocytes and macrophages that are immunoreactive for IL-1β and TNF-α. ⁹ These cytokines are produced by activated macrophages, and they may be responsible for astrocytic activation because interleukin (IL)-6, IL-1β and tumor necrosis factor-α activate astrocytes. ²⁶

FIP pathogenesis has not been fully elucidated, but FIPV infection and replication in monocytes and the activation of these cells presumably play an important role in the induction of vasculitis and granulomatous lesions in cats with FIP. ² FIPV can enter the CNS through infected macrophages, and there is no evidence that intrinsic cells of the neuroparenchyma, such as astrocytes, are infected with the virus once it enters the CNS. ²⁷ In the present study, FIPV antigens were mainly detected within the cytoplasm of mononuclear cells morphologically consistent with macrophages. However, within the brain there are several types of myeloid derived cells, including microglial cells (resident macrophages), dendritic cells and perivascular, meningeal and choroid plexus macrophages. ²⁸ Therefore, the role of these cells in FIP neuropathogenesis still remains to be established.

Conclusions

In cats with severe lesions within the neuroparenchyma due to FIPV infection there is an important activation of astrocytes which may partially contribute to the development of degenerative and inflammatory changes in the CNS. These lesions were characterized by the presence of a high number of less differentiated vimentin-positive astrocytes and a large number of gitter cells positive for RCA-1.