Hassan, Zeinb A.; Abd El-Haleem, Manal Reda; Amer, Mona G.
Interferons (IFNs) are a man-made copy of a substance that some types of white blood cells formulate naturally in the body as part of the immune response, when the body reacts to an infection or to cancer . IFNs are classified as helical cytokines and are categorized as type I or type II according to their physical and functional properties. Type I IFNs are further divided into α (leukocytes), β (fibroblasts), τ, and ω subtypes that likely diverged from a common ancestral gene .
IFN-α is used therapeutically because it has the ability to induce an ‘antiviral’ state in cells, inhibiting cellular proliferation and immunomodulation. Several preparations of both natural and recombinant IFN-α have been approved by the Food and Drug Administration for the treatment of chronic hepatitis B and chronic hepatitis C virus (HCV) and to treat several different types of cancer, particularly renal cell cancer, malignant melanoma, multiple myeloma, and some types of leukemia . Approximately 123 million individuals worldwide are infected with the HCV, surpassing the human immunodeficiency virus five-fold . Egypt has a high prevalence rate of HCV infection and as much as 90% of its genotype is type 4 .
IFN-α-2a was effective for the treatment of patients with genotype 4 HCV . However, IFN-α produces adverse effects including pathologic autoimmune effects . The most common adverse events associated with IFN-α therapy are flu-like symptoms, leukopenia, thrombocytopenia, depression, and thyroid disorders . Several studies have linked IFN-α therapy to a high incidence of retinopathy, indicated by the presence of cotton wool spots, hemorrhage, macular edema, and thrombotic microangiopathy. Although these complications are rare, some are irreversible [9,10].
α-Lipoic acid (ALA) is a very important cofactor for mitochondrial enzymes. It has been shown to be a novel biological antioxidant that destroys many of the free radicals in both the fatty and the watery regions of cells. An exogenous administration of this agent has been shown to have therapeutic potential in neurodegenerative disorders . ALA is often used to prevent ischemic optic nerve damage in glaucoma. It is also used to enhance color visual fields and visual sensitivity. Pretreatment with ALA has been found to protect the optic nerve from damage by cyanide, glutamate , and diabetes [13,14].
The aim of this study was to assess the structure of the optic nerve after IFN-α therapy and to determine the possible role of ALA in IFN-induced optic nerve complications.
Materials and methods
Commercially available injectable IFN-α-2a (Reiferon; Minapharm, Cairo, Egypt) 3 MIU/ml was used. ALA (light yellow powder, Code No. T5625; Sigma-Aldrich, Saint Louis, USA) was used. Both drugs were dissolved in phosphate-buffered saline.
This study included 40 adult male albino rats weighing (150–200g) that were held in stainless-steel cages and maintained at room temperature. They were allowed water ad libitum and were fed a standard laboratory diet. Animals were kept for 14 days before beginning the experiment for acclimatization.
The rats were divided equally into four groups: I, II, III, and IV.
Group I: was the control group, which was subdivided into two equal subgroups, Ia: five rats received phosphate-buffered saline as a vehicle and Ib: five rats received no treatment.
Group II: (ALA group) included rats that received ALA alone.
Group III: (IFN-α group) included rats that received IFN-α alone.
Group IV: (combined group) included rats that received both IFN-α and ALA.
Rats of groups III and IV received IFN-α at a dose of 100000 IU/kg three times a week, which is considered a normal treatment dose . Rats of groups II and IV received ALA at a dose of 100mg/kg once daily .
All the test substances were delivered through an intraperitoneal injection three times a week for IFN-α and once daily for ALA. At the time of sacrifice after 8 weeks, each rat was deeply anesthetized with ether, the thorax was opened, a cannula was placed in the left ventricle, the descending thoracic aorta was clamped, and the right atrium was opened. Through the cannula, perfusion with 4% glutaraldehyde in 0.9% saline was initiated. The perfusion was stopped when the venous return from the right atrium became clear . Eyes and optic nerves were rapidly enucleated. Optic nerves were dissected from 1mm behind the sclera to the optic chiasm, resected, and prepared for light and electron microscope examinations.
For light microscope examination, optic tissues were fixed in 10% neutral formal saline; they were processed into 5μm thick paraffin sections for H&E stain  and immunohistochemical staining for the detection of glial fibrillary acidic protein (GFAP) as an indicator for astrocyte reactivity . An immunohistochemical reaction was carried out using an avidin biotin peroxidase system. The primary antibody used was mouse monoclonal antibody obtained from Sigma-Aldrich (Code No. 00145904). The universal kit used the avidin biotin peroxidase system manufactured by NovaCastra Laboratories Ltd, Newcastle upon Tyne, UK. The same method was applied to prepare negative control sections but the primary antibody was not added. Mayer's hematoxylin was added as a counter stain. Rat brain was used as a positive control tissue.
Specimens for the electron microscope were immediately fixed in 2.5% glutaraldehyde buffered with 0.1mol/l phosphate buffer at a pH of 7.4 for 2h and then postfixed in 1% osmium tetroxide in the same buffer for 1h. They were processed to prepare semithin sections and then ultrathin sections. Semithin sections (1 mm thick) were stained with 1% toluidine blue for light microscope examination . Ultrathin sections were obtained using a Leica ultracut (Leica Ltd, Glienicker, Berlin, Germany) and stained with uranyl acetate and lead citrate . They were examined using a JEOL JEM 1010 electron microscope (Jeol Ltd, Tokyo, Japan) in the Electron Microscope Research Laboratory of the Histology and Cell Biology Department, Faculty of Medicine, Zagazig University, and a JEOL JEM 1200 EXII electron microscope (Jeol Ltd) in the Electron Microscope Research Laboratory of the Faculty of Science, Ain Shams University, Egypt.
For morphometric measurements, area % of positive reactions in GFAP-immunostained sections was estimated using the ‘Leica Quin500C’ image analyser computer system (Leica Imaging System Ltd, Cambridge, UK). The procedure was performed using immunostained sections at a total magnification of × 400 and measured in five different nonoverlapping fields from randomly chosen five animals from each group. Image analysis was carried out at the Histology Department, Faculty of Medicine, Cairo University, Cairo, Egypt.
Analysis of data
Statistical differences between the groups were tested by analysis of variance and the least significant difference test of the SPSS statistical package (version 17; Chicago, Illinois, USA). Data were presented as mean ± SD, and P values less than 0.05 were considered as significant.
Histological examination of the subgroups Ia and Ib of the control group and group II (ALA group) showed no differences; thus, we use figures (control, subgroup Ia) to represent them.
Group I (control group Ia)
Light microscope examination of the sections of the optic nerve of the control adult male albino rats showed that the optic nerve was composed of multiple adjacent closely packed nerve axons (Fig. 1). Most of the optic nerve axons were myelinated. They were compactly arranged and variable in diameter. The axons appeared as clear areas with a dark ring of myelin around them. The nerve fascicles were surrounded by a thin perineurium. Nuclei of neuroglial cells were scattered in between the axons (Fig. 2). A few GFAP immunepositive astrocytes with short thin cytoplasmic processes were detected (Fig. 3). Electron microscope examination of the ultrathin sections of the optic nerve of the same group showed that myelinated axons had varied diameters. A myelin sheath was composed of compact lamellae surrounding the axoplasm. Their axoplasm contained mitochondria, neurotubules, and neurofibrils. Oligodendrocytes had euchromatic nuclei and many cytoplasmic processes that encircled many axons (Fig. 4). The capillary wall was composed of a thin layer of an endothelial cell cytoplasm and a clear basement membrane (Fig. 5).
Group III (the group that received IFN)
Light microscope examination of the sections of the optic nerve of the rats that received IFN showed that the optic nerve had many neuroglial nuclei compared with the control group (Fig. 6). Nerve axons were loosely arranged. The perineurium was thickened compared with the control (Fig. 7). Extensive expressions of GFAP immune reactive astrocytes with long cytoplasmic processes were observed (Fig. 8). Electron microscope examination of the ultrathin sections of the optic nerve of the group that received IFN-α showed disorganization of different-sized nerve fibers. Most of the myelin sheaths showed alterations in their morphology, such as split myelin layers and thinning. A few nerve axons still retained their normal structure. Oligodendrocytes had a vacuolated cytoplasm (Fig. 9). Some mitochondria in the axoplasm appeared swollen with destroyed cristae (Figs 9 and 10). Some neuroglial cells were observed to have an electron-dense small nucleus, a dense cytoplasm, and shrunken cellular processes. Other neuroglial cells had nuclei with peripheral heterochromatin (Fig. 11). Some axons were shrunken and showed axolemmal detachment from distorted myelin (Fig. 12a). A few axons showed splitting of their myelin into multilayered whorled masses, forming myelin figures (Fig. 12b–14). Some axons were swollen pale. They showed axolemmal expansion, and a few neurotubules and neurofibrils (Fig. 13). Extravasated red blood cells were observed between distorted nerve axons. Many blood capillaries had distorted, ill-defined walls (Fig. 14). Most of the blood capillaries were lined by endothelial cells with irregular heterochromatic nuclei that protruded into the lumen. Their basement membranes were ill-defined. Bundles of collagen fibers surrounded the blood capillaries (Fig. 15).
Group IV (combined group)
Light microscope examination of the sections of the optic nerve of the rats that received both IFN-α and ALA showed that the optic nerve had almost retained its normal architecture (Fig. 16). Many nerve axons of variable diameters appeared to be compactly arranged. Neuroglial nerve cells had pale nuclei (Fig. 17). Decreased GFAP immunoreactive astrocytes with thin cytoplasmic processes were observed (Fig. 18). Electron microscope examination of the ultrathin sections of the optic nerve of the same group showed an apparently normal optic nerve. It was composed of multiple tightly packed myelinated axons of variable diameters. Their axoplasm contained neurofibrils and mitochondria with an almost normal appearance and were surrounded by multiple compact myelin layers. Astrocytes appeared with a prominent euchromatic indented nucleus. Some destroyed axons could still be observed (Fig. 19). A few myelin sheaths appeared to have been disrupted with compact lamellated areas and homogenous areas. Some oligodendrocytes had heterochromatic nuclei (Fig. 20). Blood capillaries appeared almost normal. They were surrounded by well-defined basal lamina (Fig. 21).
Morphometric and statistical results
Area % of positive reactions in GFAB-immunostained sections showed a nonsignificant difference between subgroups Ia and Ib of the control group and group II (ALA). Area % of positive reaction in GFAB-immunostained sections showed a significant increase in the group that received IFN-α (group III) in comparison with the control group (group I), whereas a significant decrease in area % of GFAP-positive immunreactions was observed in the combined group (group IV) in comparison with the group that received IFN-α (group III) (Table 1 and Histogram 1).
IFN-α is commonly used for the treatment of patients with chronic viral infection and malignant disorders . Long-term treatment is required, thus increasing the risk of side effects including ophthalmic complications . In a prospective series, 70% of HCV patients treated with IFN developed retinopathy that was transient or occurred in recurrent episodes , with little data on the effects on the optic nerve.
Rodent optic nerve is a typical central nervous system white matter tract that contains axons of retinal ganglion cells together with the glia that support them, namely astrocytes, oligodendrocytes, and microglia . The absence of neuronal cell bodies and synapses indicates that the optic nerve is an anatomically simple tissue for studying the physiology and pathophysiology of central nervous system white matter and axonal–glial interactions . Therefore, the aim of this study is to study the histological structure of rat optic nerve after an injection of IFN-α and to determine the role of ALA supplementation.
In this study, examination of the optic nerve of rats that were administered IFN showed nerve damage manifested as variable degrees of degeneration. The optic nerves of the IFN-treated group showed loosely arranged, disorganized, and different-sized nerve fibers separated by thick perineurium. Degeneration of the myelin in the form of thin, denuded, and irregular whorls of myelin forming myelin bodies was observed. Some axons were swollen and appeared pale and enlarged with axolemmal expansion and cytoskeletal disintegration. Others had degenerated swollen mitochondria with disrupted cristae. Similar effects on the optic nerve have been reported after IFN-α therapy  and after the administration of other toxins . It is unclear whether the same axons show different types of axoplasmic degeneration at variable distance from retinal ganglion cells at the same time or whether individual axons undergo a specific type of degeneration throughout its length . Swollen axons appeared pale and enlarged with cytoskeletal disintegration and axolemmal expansion. Similar structural changes were detected in optic nerve, was detected in optic nerve during excitotoxic Wallerian degeneration .
In the current study, splitting of myelin into multilayered whorled masses that formed myelin figures was observed in the group that received IFN. Similar finding was named as myelin bodies, Which, were formed as a result of phagocytic action of astrocytes and microglial cells which were scattered in the extracellular space. These cells invade the myelin sheath and phagocytose the peeled-off outer lamellar myelin debris. Phagocytosed myelin decomposes and forms a homogeneous or a heterogeneous osmophilic layered structure called a myelin body, which finally disintegrates and transforms into globoid lipid droplets and needle-shaped cholesterol crystals .
Thin or denuded myelin in the group that received IFN could have occurred secondary to the retrograde loss of axons initiated by the loss of retinal fibers. Another possibility is that IFN-α leads to anterior ischemic optic neuropathy (AION). The exact mechanism of this is unknown ; AION is an optic nerve stroke and leads to progressive stress of oligodendrocytes, which ultimately make them dysfunctional. Thus, myelin degeneration and loss occurred [7,29]. Patients with AION typically present with sudden, painless loss of vision associated with swelling of the optic nerve in the affected eye, followed by disruption of the normal nerve architecture . Some authors have proposed that therapy with IFN-α may result in autoantibody formation and immune complex deposition, with the resultant lymphocyte infiltration and inflammation of vessels, leading to optic nerve ischemia .
In the current study, degeneration of glial cells was observed after the administration of IFN. Oligodendrocytes were observed to have heterochromatic nuclei, a vacuolated cytoplasm, and disintegrated mitochondria. Others had dense small nuclei, a dense cytoplasm, and shrunken cellular processes. Oligodendrocytes are essential for neuronal cell body and axon survival  as well as for myelin assembly . The current findings of oligodendrocytes are in agreement with other studies that showed that oligodendrocytes are susceptible to ischemia and rapidly become dysfunctional [32,33]. Oligodendrocytes are highly susceptible to oxidative stress because of their low antioxidative defense systems and high metabolic rate. Thus, oxidative stress is considered to play an important role in optic nerve affection .
In the current study, after IFN treatment, astrocytes underwent reactive changes and this was confirmed by the extensive expression of immune positive GFAP astrocytes with long cytoplasmic processes. These responses to IFN treatment appeared to be a result of adaptive processes countering neurotoxic effects in a manner similar to that observed during Wallerian degeneration . Astrocytes play many roles that are critical for normal optic nerves function; they contribute to the blood–optic nerve barrier as they form webs between the vascular system and axons in the optic nerve. They also participate in the scarring and repair of the nervous system . The current change in the astrocytes is known as reactive astrogliosis. Astrogliosis could be a result of the stimulation of the mitotic activity of astroglia by a neuritic injury . Fibroblast growth factor was released from macrophages in the area of neuritic injury into the surrounding . It has been reported that glioblasts (glial precursor cells) are common precursors of oligodendrocytes and astrocytes . This may preferentially stimulate astrocyte rather than oligodendrocyte division, leading to glial scarring and a subsequent failure of remyelination. Thus, these large numbers of astrocyte cells that are present after an IFN insult could be because of the proliferation of glioblasts .
In the current study, signs of inflammation of the optic nerve such as axonal swelling, astrogliosis, and extravasated red blood cells were found in the group that received IFN. Such changes in the optic nerve could play two contrasting roles. The inflammation can cause increased tissue destruction, edema, and compression of adjacent vessels and axons . However, macrophages play a major role in phagocytosis and the removal of myelin debris after ischemic axonal degeneration to enhance the potential for remyelination . Therefore, selective manipulation of this inflammatory response may represent a new treatment for ischemic optic nerve diseases .
In the present study, the optic nerve of the group that received IFN showed distorted blood capillaries. Their basement membranes were ill-defined. Bundles of collagen fibers surrounded the blood capillaries. Their endothelial cells contained indented heterochromatic nuclei that protruded into lumina. Similar effects were found with IFN on the optic nerve  and other cranial nerves . Some investigators have proposed that the antiviral agent deposits immune complexes in the optic vessels, causing ischemic changes . Other studies hypothesize a process similar to diabetic neuropathy in which the microvasculature is damaged, leading to ischemia . As an immunomodulator, IFN-α stimulates other cytokines, such as interleukins. These interleukins may cause an inflammatory reaction of the blood vessels, leading to ischemia .
The process of ischemia itself causes no change in nerve permeability, but the subsequent reperfusion results in increased permeability of the blood–nerve barrier, an increase in nerve lipid hydroperoxides, and an increase in endoneural edema. Reperfusion results in a burst of free radicals during reoxygenation of hypoxic tissue .
In this study, it was observed that the concomitant administration of ALA with IFN resulted in less degenerative changes than those observed with the administration of IFN alone. The optic nerve retained its normal architecture. Only a few nerve fibers showed degenerative changes in the form of homogenous areas in their myelin sheath alternating with compact lamellated areas. Blood capillaries were apparently normal. Many studies that have studied the protective role of ALA in nervous tissue have shown that ALA provides protection to the retina as a whole, and to ganglion cells in particular, from ischemia–reperfusion injuries  and optic neuritis . ALA could induce an increase in cell viability, a decrease in intracellular protein turnover, and a decrease in oxidant-induced protein oxidation. Therefore, the excessive oxidative damage of oligodendrocytes and their protein pool can be prevented by the use of ALA . In agreement with the previously reported results, some investigators have found that ALA decreases reactive oxygen species generated by redox cycling of menadione and to generate nitric oxide. Therefore, it could be suggested that ALA enhances both the antioxidant defenses and the function of endothelial cells. This can explain its protective role in blood capillaries .
In conclusion, the results of this study showed that the IFN induced marked structural changes in the optic nerve of albino rats. These changes could explain the ophthalmic complications and visual impairment associated with IFN therapy. Moreover, ALA provided significant protection against these changes, which was confirmed morphometrically and statistically. Thus, ALA could be used as a promising therapy in neuroprotection strategies. We recommend further areas of research focused on the mechanisms of neurodegeneration following IFN-induced injury to the optic nerve and clinical studies to explore the effects of long-term administration of ALA.
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Conflicts of interest
There is no conflict of interest to declare.
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