Multiple sclerosis (MS) is considered a chronic inflammatory disease of the central nervous system (CNS), characterized by multifocal, disseminated inflammation, demyelination, axonal damage, and gray matter pathology 1–4. To this end, experimental autoimmune encephalomyelitis (EAE) has been established as an animal model for MS 5–10. EAE can be induced either by active immunization with CNS antigens or by the passive transfer of encephalitogenic T cells into susceptible animal strains. In rat models, a pattern of axonal damage and neuronal injury is seen after immunization with myelin oligodendrocyte glycoprotein 11. This model is similar to many aspects of MS. After immunization, axonal damage was observed in the white matter of monkeys 12 and in focal EAE models of rat cortices. Some other authors have delineated CNS inflammation, demyelination, axonal damage, and motor neuron atrophy among the side effects induced by myelin oligodendrocyte glycoprotein peptides 13,14. EAE models not only allow investigation of axonal injury but also help to find ways of recovery and axonal protection. In a study using a focal rat EAE model, remodeling of axonal connections could be investigated 15,16. Some earlier studies in rodents characterized neuroprotective treatment approaches like blocking of glutamate receptors 17. However, all previous studies relied on light microscopic analysis, which could give only a general overview of the pathologic traits of the model, falling short in providing details about the fine characteristics of nerve fiber and neuronal damage 18,19.
Aim of the work
The aim of the study was to investigate the histological pattern of axonal injury and the mechanism of demyelination in autoimmune encephalomyelitis in the cerebral cortex and spinal cord of adult albino rabbits.
Materials and methods
Twenty adult male albino rabbits (2.5–3.0 kg) obtained from the animal house, Moshtohor Faculty of Veterinary Medicine, Benha University, were used in the experiment. All rabbits received a balanced diet with free access to water. All animal procedures were performed according to approved protocols and in accordance with the recommendations for the proper care and use of laboratory animals. The animals were divided into two groups: group I was considered the control group (10 rabbits). In group II (the experimental group; 10 rabbits), each rabbit was injected with 0.3 ml/kg of 0.01% ethidium bromide solution intrathecally to induce encephalomyelitis 20.
Induction and clinical assessment of experimental autoimmune encephalomyelitis
Experimental encephalomyelitis was induced by the injection of 0.3 ml/kg of 0.01% ethidium bromide solution (Sigma, Cairo, Egypt), which was a modification of the dose used in the study by Pereira et al. 20. We reduced the dose by 0.1 ml/kg every time until we reached a dose of 0.3 ml/kg, which was the dose used in this study to avoid death of the animal. The animals were injected intrathecally by exposing the thecal fosse under general anesthesia (administered using 90 mg ketamine and 10 mg xylazine/kg) and aseptic conditions. Evidence for the presence of encephalomyelitis was tested clinically by assessing the motor activity of the animals. Positive cases showed quadriplegia.
Two weeks after intrathecal injection of ethidium bromide the animals were sacrificed and the white matter of the frontal lobe of their cerebral cortex and spinal cord was processed for light microscopic examination. In addition, an electron microscopy examination was performed on the spinal cord.
The rabbits were perfused intracardially with 4% paraformaldehyde/4% glutaraldehyde in 0.1 mol/l PBS (pH=7.4). The spinal cords were removed carefully from the vertebral canal. The lumbosacral part was cut off using a sharp blade. The tissue was postfixed at 4°C for at least 24 h. The specimens were rinsed in cacodylate buffer three times and treated with 1% osmium tetroxide for 4 h on ice. After repeated rinsing, the tissue was dehydrated in a graded series of ethanol and treated with 1% uranyl acetate in 70% ethanol for contrast enhancement overnight. Subsequently, the specimens were embedded in epon. Thin sections of each plastic-embedded spinal cord sample were cut on an ultramicrotome (Reichert, Bensheim, Germany). The ultrathin sections were stretched with xylene vapors and carefully suspended on 150 mesh hexagonal Formvar-coated copper grids. The preparations were stained with 1% aqueous uranyl acetate solution for 20 min and Reynold’s lead citrate solution for 7 min. The sections were examined using a transmission electron microscope, and images were taken with an electron microscopy digital camera system (MegaView, analysis docu 3.2; Olympus Soft Imaging Systems GmbH, Münster, Germany).
Sections were measured using the Olympus BX40, DOT Med’s Shipping Quote Service, UPS, (USA), image analyzer computer system at the Histology Department, Faculty of Medicine, Cairo University. Measurements were taken within 10 nonoverlapping fields for each animal at ×400 magnification with H&E stain. The Student t-test calculated with Sigma Stat software (version 7.0; SPSS, Chicago, Illinois, USA) was used to count the number and mean surface area of pyramidal cells and motor neurons and the mean surface area of their nuclei. Statistical significance was set at P values greater than or equal to 0.05.
Light microscopic result
The cerebral cortex of the control group showed neuronal layers: an outer molecular layer (I), an external granular later (II), an external pyramidal layer (III), an inner granular layer (IV), and inner pyramidal (V) and polymorphic layers (VI) (Fig. 1).
Examination of the cerebral cortex showed pyramidal cells with pale nuclei and prominent nucleoli. The cells had apical dendrites, neuropils consisting of nerve fibers, neuroglial cells, and blood vessels (Fig. 2).
Examination of a section of the spinal cord in group I showed central gray matter and peripheral white matter. The gray matter contained nerve fibers, nerve cells, and neuroglial cells. Many polygonal motor cells with many processes, central vesicular nuclei, and Nissl’s granules could be seen. The white matter contained thick fibers and neuroglia cells. The nuclei of oligodendroglia could be seen arranged in rows between the nerve fibers (Fig. 3).
A higher magnification of the white matter in group I showed many nerve fibers (cut transversely), blood vessels, and neuroglia (Fig. 4).
The cerebral cortex of group II rabbits showed subpial and perivascular cellular infiltration extending into neuropil tissues (Figs 5 and 6). The pyramidal cells of the cerebral cortex showed thick, tortuous cellular processes with darkly stained nuclei (Fig. 7). Some areas of the cortex showed necrosis of some nerve cells and neuroglia (Fig. 8). The spinal cord of group II showed swelling of some cells in the grey matter. The nucleus became eccentric with loss of cellular processes and Nissl’s granules. Some areas showed cellular aggregation (Figs 9 and 10).
Electron microscopic results
Ultrastructurally, the anterior horn motor neurons of the spinal cord were easily identified by their large size and characteristic shape. The nuclei were centrally located, rounded to oval in shape, and euchromatic. The nucleoli appeared large (Fig. 11).
Degeneration is a consistent feature of experiment-induced experimental autoimmune encephalomyelitis
The axons in the white matter of the spinal cord of the control group showed microtubules, microfilaments, and mitochondria and were surrounded by a myelin sheath (Fig. 12).
The gray matter in the spinal cord of group II showed degenerated nerve cells (Fig. 13). The white matter of the spinal cord showed several areas of degenerated nerve fibers and many astrocytes (Fig. 14). Many axons showed splitting of the myelin sheath (onion bulb appearance) (Fig. 15), whereas others showed defective myelination in the form of a thin or absent myelin sheath (Fig. 16).
Morphometrical and statistical results
There was significant decrease in the mean number and mean surface area of pyramidal cells and mean surface area of their nuclei in the experimental group in comparison with the control group (Histogram 1 and Table 1).
The present study showed mononuclear inflammatory cells, especially subpial and perivascular, and in the neuropils of the cerebral cortex. Similar findings were found by other investigators as well 21. Inflammation was associated with apoptotic loss of some neurons and neuroglia cells in the cerebral cortex of the present study as well as in others 22. Motor neurons of the cerebral cortex of the present study showed hyperchromatic nuclei with many thick and tortuous processes. Some scientists 23 explained that this thickness might be due to accumulation of phosphorylated neurofilaments in the cell process resulting in impaired axonal transport. This might result in degeneration and death of neurons. Meanwhile, other scientists recorded that increase in nerve cell process might represent attempted regeneration of the axons in the CNS 24,25. Moreover, motor cells of the spinal cord of the present study became swollen with withdrawal of their processes. Their cytoplasm showed eccentric nuclei and absence of Nissl’s granules. These changes might be due to axonal injury, as seen in the present work. Massive loss of axons was associated with inflammation 26.
In contrast, some investigators recorded that axonal injury occurred independent of inflammation 27. Moreover, some scientists recorded re-expression and redistribution of sodium channels at the site of the axonal injury. This re-expression constituted an attempt to maintain conduction in demyelinated axons. Redistribution of the sodium channel caused a change in the intracellular sodium levels. This led to reverse action of Na and Ca exchanges and to an increase in Ca overload. This caused activation of certain proteases that destroyed the cytoskeleton of the axon 28.
Defective myelination in the form of splitting thinning and loss of myelin sheath was recorded in the present work 1.
In the present study white matter (common site of demyelination) was examined using an electron microscope. The present study revealed many astrocytes and oligodendroglia in the white matter of the spinal cord. Normally, white matter of the spinal cord contained few glial cells compared with gray matter 29.
The present study has shown that axonal damage in the white matter is a significant contributor to the disability seen in the EAE model.
Conflicts of interest
There is no conflict of interest to declare.
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Keywords:© 2013 The Egyptian Journal of Histology
cerebral cortex; spinal cord; encephalomyelitis; rabbit