Apoptosis in Natural Rabies Virus Infection in Dogs
Apoptosis in Natural Rabies Virus Infection in Dogs
OPEN DOI:10.1515/jvetres-2016-0034
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Abstract
Introduction: In the present study apoptosis was investigated in the cornu ammonis and cerebellum of 10 dogs naturally
infected with rabies virus. Diagnosis of rabies was based on the results of fluorescent antibody staining and experimental
inoculation. Material and Methods: The paraffin tissue sections were stained with haematoxylin and eosin, avidin-biotin
complex peroxidase (ABC-P), and terminal deoxynucleotidyl transferase biotin-dUTP nick end-labelling (TUNEL) methods.
Results: Histopathological examination revealed encephalomyelitis of varying severity and the presence of Negri bodies. Dense
rabies antigens were determined in the motor neurons with ABC-P method. On the other hand, Bcl-2 protein and Bax protein
gave positive reaction in seven and five cases, respectively. TUNEL staining demonstrated very marked apoptotic changes in the
nuclei of neurons localised deep in the substantia alba of the cerebellum. Similar changes were also determined in perivascular
mononuclear cells and glia cells within the substantia alba. No apoptopic changes were found in the motor neurons of the cornu
ammonis. Conclusion: The absence of apoptotic changes in the neurons was considered to be the consequence of the necrotic
changes that developed in these neurons.
© 2016 S.A. Vural et al. This is an open access article distributed under the Creative Commons
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22 S.A. Vural et al./J Vet Res/60 (2016) 227-
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mechanism of cells and results in the fractures of the the samples were incubated with AEC and
DNA helix. It starts with the stimulation of some counterstained with Gill’s haematoxylin. As control,
internal and external factors. There are biochemical and the samples were treated with labelling solution instead
morphological changes in the stimulated cells (6). of TdT.
These reactions are formed directly or indirectly by an The severity of immunoreactivity was graded
increase in the level of substances that are under according to the density in 10 different microscopic
control of apoptotic genes (Bax, Bad, Bad protein- areas as + mild, ++ moderate, +++ severe, - no staining.
proapoptotic) or anti-apoptotic genes (Bcl-2, Bcl-xl,
Boa). All these substances and energy required for
apoptosis are supported by mitochondria (7, 20).
Results
In these days, the role of apoptosis has been
studied in regard to experimental infections of mice
Histopathologically, many of the vessels in the
with Sindbis virus, Crosse virus, and reovirus (4, 10,
cornu ammonis and cerebellum were hyperaemic and
14, 16). The present study describes the distribution of
surrounded by mononuclear cells. Similar perivascular
viral antigen and apoptosis in the central nervous
cells were also seen around meningeal vessels. Some
system of dogs infected with street rabies virus.
neurons were degenerative or necrotic. Neuronophagia
and Babes’ nodules were visible. The proliferation of
glia cells was focally observed and different numbers
Material and Methods of Negri bodies were seen in all samples (Figs 1-3).
Morphological changes related to apoptosis also drew
Animals. The study involved 10 dogs which had attention, especially in internal/external granular layer
been determined to be rabies-positive based on the (Fig. 4) and at cerebellar nucleus in the cerebellum, to
results of fluorescent antibody staining and a lesser extent in the neurons located in the cornu
experimental inoculation. ammonis. The most evident feature of these changes
Sample collection and histopathological was the phenomenon of neurons becoming smaller and
examinations. The cerebellum and cornu ammonis shrinking, cytoplasm getting narrow, and chromatins
were fixed in 10% buffered formalin, processed by coming together as rough particles near the nuclear
routine method, and embedded in paraffin. The 5 μm membrane.
sections were mounted on glass slides, and stained with Immunohistochemically, high levels of rabies
haematoxylin and eosin (HE). Selected sections were virus antigen were detected in motor neurons of the
also stained with cresyl violet. cornu ammonis, Purkinje cells, external layer, and
Immunoperoxidase staining. The sections were cerebellar nucleus (Fig. 5) in all animals. Staining of
stained for rabies virus antigen by avidin-biotin Bcl-2 protein was positive in seven dogs and Bax
complex peroxidase (ABC-P) method using polyclonal protein in five animals (Figs 6-9). Bax protein was
rabbit anti-rabies virus nucleoprotein sera (1:500, detected in the Purkinje cells, cerebellar nucleus, and
Animal Disease Research Institute, Canada) and external granular layer of the cerebellum contrary to the
polyclonal rabbit anti-Bax and Bcl-2 antibodies (1:500, cornu ammonis. On the other hand, Bcl-2 protein was
Santa Cruz Biotechnology, USA) according to the localised in the Purkinje cells, cerebellar nucleus, and
procedure provided by the producer (LSAB Kit, Dako). motor neurons of the cornu ammonis.
The colour was developed by a final incubation with 3- At TUNEL staining, the most evident reaction was
amino-9 ethylcarbazole (AEC). As a control group, the found in external/internal granular layer and cerebellar
samples were treated with normal rabbit sera instead of nucleus of the cerebellum. Similar changes were seen
primary antibody. in perivascular mononuclear cells (Fig. 10) and glia
In-situ detection of apoptotic cells by TUNEL cells which were located in substantia alba. At the same
assay. DNA fragmentation was assessed in situ in time, the differences in localisation and degree of
sections using the terminal deoxynucleotidyl positive reaction were not significant in the cerebellum
transferase (TdT)-mediated dUTP-digoxigenin nick of all dogs. No positive TUNEL reactions were seen in
end-labelling (TUNEL) kit as recommended by the the cornu ammonis.
manufacturer (In Situ Cell Death Detection Kit, POD, The results are summarised in Tables 1 and 2.
Roche, Germany). To visualise the reaction products,