Lesions included neuronal damage characterized by chromatolysis, increased cytoplasmic eosinophilia, nuclear shrinkage and in advanced stages scavenging by microglia (microgliosis). In addition, axonal damage was found located most prominently in the pons, especially in central auditory and vestibular pathways of the medulla oblongata, in cerebellar nuclei and nucleus pontis.
In experimental group no. 7 the most frequently observed alterations in the “Gray matter group no. 1” were central chromatolysis (23.72%), and total chromatolysis (22.79%). A reactive change, such as microgliosis (11.63%) was considered as a
response to the neuronal damage. In “Gray matter group no. 2” the most commonly observed lesions were central chromatolysis (27.84%), total chromatolysis (25.77%), eosinophilic cytoplasmatic granulation (15.46%), and microgliosis (10.31%). The
“White matter group no. 1 and 2” revealed few damaged axons located predominantly in the corpus trapezoideum and tractus spinalis nervi trigemini In experimental group no. 8 the most frequently observed alterations in “Gray matter group no. 1” were total chromatolysis (27.91%), central chromatolysis (24.42%), axonal damage (spheroids) (10.47%), and reactive changes including microgliosis (8.14%). In “Gray matter group no. 2” the most frequently observed lesions were total chromatolysis (31.43%), central chromatolysis (26.57%), eosinophilic cytoplasmatic granulation (14.29%), and vacuolization of the neuropil (10.31%). “White matter group no. 1” was normal. However, the “White matter group no. 2” showed a few damaged axons located predominantly in the fasciculus retroflexi. The other experimental groups revealed few histopathological changes including central chromatolysis that is considered as a normal finding in some nuclei including nucleus olivaris, nucleus pontis, and nucleus supraopticus (SUMMERS et al., 1995). In addition, minimal axonal damage as an idiopathic lesion in different areas of the brain stem as well as little inflammatory changes in leptomeninx and choroid plexus were present. Artemether (ARM566) with dose of 40 mg/kg administrated intramuscularly for 8 days induced a neurono- and axonopathy as well as some reactive changes including inflammatory response, gliosis and microgliosis restricted selectively to certain brain areas.
5.3.1 Chromatolysis
Total chromatolysis was observed as significant finding only in dogs treated with high doses of (ARM566) for 8 days intramuscularly (experimental group no. 7 and 8). The other experimental animals and the vehicle control lacked total chromatolysis.
In addition, central chromatolysis was observed in experimental group no. 7 and 8 more frequently than in other groups. This observation may indicate that central chromatolysis appears to be the primary response of a neuron to the toxic effect of both artemisinin formulations. Most likely, total chromatolysis may develop subsequently. Central chromatolysis was found in treated and untreated groups
especially in the nucleus olivaris anterior and nucleus olivaris posterior, which is considered as a normal finding in these localizations (SUMMERS et al., 1995).
Chromatolytic neurons of the brain stem were visualized using LFB stain for a more precise demonstration of the distribution of Nissl’s substance. The different frequency of chromatolytic neurons in H&E- and LFB-stained sections was most likely due to different cutting levels and the higher sensitivity of the LFB stain to Nissl’s substance.
The latter is due to a better outlining of swollen chromatolytic neurons compared to H&E-stained sections.
Most of the recent experimental studies have reported that neuronal cell damage including chromatolysis is an effect of artemether in dogs, rats (BREVER et al., 1994), and in mice (NONTPRASERT et al., 2002)
The amount of Nissl’s substance depends on the physiological state of the cell and is concerned with protein synthesis and metabolism with variable physiological and pathological conditions (ANDREW, 1936). The pathogenesis of chromatolysis in artemisinin treated animals is not completely understood until now, but the lesion may occur due to a direct toxic effect on the neuron or its microstructure.
Alternatively, chromatolysis may develop as response to a metabolic disturbance of a neuron after drug administration (LEVINE et al., 2004). Chromatolysis may occur also as a response to damage of its peripheral processes (ANDREW, 1936).
The histological findings of chromatolysis were highlighted using immunohistochemistry with antibodies for neurofilaments. Antibodies specific for p-NF identify phosphorylated epitopes of neurofilaments. These antibodies immunostain exclusively axons and dendrites in normal neuroparenchyma and do not react with perikarya. Approximately 80% of the axonal NF is phosphorylated (NIXON, 1993). Antibodies specific for n-NF react with non-phosphorylated epitopes of neurofilaments, which are located exclusively in the perikarya of normal neurons.
In present study chromatolytic neurons, which were the hallmark of artemether neurotoxicity in affected beagle dogs, showed an expression of p-NF in their perikarya. Conversely, the perikarya of not-affected neurons in all other animals were negative for p-NF. This expression suggests, that an aberrant and perhaps noxiously phosphorylation occurred and has to be attributed to a toxic effect of the artemether applied in high doses intramuscularly. Alternatively, it has to be considered, that
phosphorylation may occur as a “non-specific” response of the perikaryon to other unknown endogenous and/or exogenous stimuli.
Neurofilament phosphorylation is supposed to be a reaction to an injury of the cell soma rather than an event related to damage and regeneration of cytoplasmic processes, which has been shown after transection of the sciatic nerve of experimental rats (MANSOUR et al., 1989). Aberrant hyperphosphorylation of neurofilaments in dendrites and cell bodies are seen in neurodegenerative diseases such as Amyotrophic Lateral Sclerosis, Alzheimer’s disease, Parkinson’s disease, Pick’s disease and Dementia with Lewy bodies (KESAVAPANY et al., 2004), and in canine distemper virus infection (SEEHUSEN, 2006). Thus, defects in compartmentalization of cytoskeletal protein phosphorylation may contribute to the pathology seen in these diseases. Neurofilament phosphorylation is affected by signal transduction pathways. Calcium influx into neurons causes the phosphorylation of NF-M through the activation of the extracellular signal regulated kinase1/2 (ERK1/2). Integrin mediated signaling also causes the phosphorylation of NF-H through the activation of Cyclin-dependent kinase 5 (Cdk5) activities. Recent studies have also shown that kinase cascades can be affected by myelin associated glycoprotein (MAG), a major glial protein found in periaxonal membranes of glial cells. MAG appears to be involved in bi-directional signaling affecting axonal properties such as axonal caliber, phosphorylation of neurofilaments and mediating the activity of ERK1/2 and Cdk5 (KESAVAPANY et al., 2004). Neurofilament proteins (NFPs) are highly phosphorylated molecules in the axonal compartment of the adult nervous system. The phosphorylation of NFP is considered an important determinant of filament caliber, plasticity, and stability. This process reflects the function of NF during the lifetime of a neuron from differentiation in the embryo through long-term activity in the adult until aging and environmental insults leading to pathology and ultimately death. NF function is modulated by phosphorylation-dephosphorylation in each of these diverse neuronal states (PANT and VEERANNA, 1995).
5.3.2 Axonal damage
Axonal spheroids were frequently found in animals of experimental group no. 7 and 8, located preferentially in the nucleus cochlearis, corpus trapezoideum (part of the auditory pathway), nucleus cuneatus externus, tractus spinalis nervi trigemini, and
fibrae vestibulo-cerebellares. This axonal damage may be caused by direct axonal injury or developed subsequently to primary somatic injury of the neuron. Axons are susceptible to various noxes (trauma, intoxication etc.) resulting in axonal injury. In the present study the cause of axonal damage remains undetermined but the significantly high prevalence of this lesion in the intramuscular high dose groups (experimental group no. 7, 8) indicates most likely a direct effect of artemether on axons and/or myelin sheaths. As differential mechanism a primary injury to the cell body with secondary axonal damage has to be considered, similar to anterior poliomyelitis or motor neuron disease (MACSWEEN and WHALEY, 1992). Affected areas especially in the central auditory pathway are either particularly susceptible to toxic effects of artemether or represent a response to neuronal degeneration or both, resulting in dysfunctions of the auditory system. However, when the axon insulation is disrupted, the speed of information processing within the brain can be profoundly affected (GRAHAM and MONTINE, 2002; KINNEY and ARMSTRONG, 2002) leading to hearing deficits, which have been observed clinically in humans in Mozambique receiving co-artemether (TOOVEY and JAMIESON, 2004). However, other studies have not shown hearing deficits (ABDULLA et al., 2008;
HUTAGALUNG et al., 2006; McCALL et al., 2006).
Axonal injury probably results in both Wallerian degeneration of the axon and/or also retrograde degeneration of the cell body due to toxic effects on both. The functional consequences of the axonal injury will depend upon numbers of axons injured and the topographical organization of the fibres coursing through the lesion. The molecular mechanisms of axonal transection are not known. However, investigations of Wallerian degeneration mutant mice with very slow Wallerian degeneration demonstrate that axon degeneration is not simply a passive disintegration of the axon but has clear parallels with the active processes of programmed cell death (PERRY and ANTHONY, 1999). The presence of early axonal injury and perhaps the consequences of an ever increasing load of neuronal damage (chromatolysis) have important implications for the therapeutic target of artemether.
In this study, affected axons (spheroids) expressed p-NF suggesting a disturbance of the axonal transport possibly caused by a direct effect of the artemether, whereas the n-NF antibody failed to label axonal spheroids. In subacute lesions of canine distemper virus infection in dogs axonal damage showed n-NF expression
(SEEHUSEN, 2006). The lack of n-NF expression in spheroids of this study suggests an acute axonal damage.
APP expression in injured axons (spheroids) appeared as finely granular labelling.
Brain APP is processed by neurons and transported by fast anterograde axonal transport (KAWAI et al., 1992; SASAKI and IWATA, 1999), therefore the failure to detect accumulated APP may be due to the lack of neuronal production of APP in the perikaryon. Alternatively, as described for other neurological disorders it may indicate an axonal damage older than 24 hours that usually lacks staining for APP (HORTOBAGYI and AL-SARRAJ 2008).
Like other axonal proteins, neurofilaments are synthesized in the neuronal cell body and transported into and along the axon by a process called axonal transport.
Considerable evidence now indicates that neurofilament triplet proteins normally exist in a hypophosphorylated form in the soma and that phosphorylation of the tail domains is a slow posttranslational modification that occurs after the neurofilament proteins have assembled into filaments and during their transit along the axon (BENNETT and DILULLO, 1985; BLACK et al., 1986; NIXON et al., 1987;
OBLINGER, 1987). Therefore it is possible that the proximo-distal gradient of neurofilament phosphorylation along axons reflects a gradient of neurofilament age, with the youngest and least phosphorylated neurofilaments located proximally, close to their site of assembly in the cell body and the oldest most extensively phosphorylated neurofilaments located distally, farest away from the cell body.
However, while neurofilament age could well be the principal determinant of the extent of neurofilament phosphorylation in insulated neurons in culture, it is likely that neurofilament phosphorylation in vivo is modulated by additional factors, such as interactions with myelinating cells (DE WAEGH et al., 1992; MATA et al., 1992;
NIXON et al., 1994; STARR et al., 1996) and possibly other environmental cues (LANDMESSER and SWAIN, 1992).
5.3.3 Gliosis
In this study gliosis was found in different experimental groups, but particularly in group no. 7. This finding suggests that these processes started as attempts to protect or repair affected neurons. Gliosis observed in H&E-stained sections was characterized by accumulation of glial cells with a morphology interpreted as
astrocytes. Glial fibrillary acidic protein (GFAP) is present only in mature astrocytes (MENET et al., 2000). However, in the present study these cells lacked GFAP-expression suggesting that they may be immature precursor stages of astrocytes or other neuroglia (supporting cells). The astrocytic response to injury proceeds through several stages and depends on the extent of injury. Approximately 4 days after injury (MAXWELL et al., 1990), there is a rapid increase in the synthesis of glial fibrillary acidic protein (GFAP) that can extend far from the actual site of damage (ABNET et al., 1991). However, many studies implicate a protective role for reactive or activated astrocytes in the post injury period of the brain (LONGHI et al., 2001). Long standing hypotheses suggest that reactive astrocytes create a physical barrier between damaged and healthy cells (FAULKNER et al., 2004). Reactive gliosis is a common universal reaction to brain injury, but the precise origin and subsequent fate of the glial cells reacting to injury are unknown.
Common pathways of neuronal cell death in response to various insults, e. g.
hypoxia, ischemia, or trauma, include early disruption of ion homeostasis, increased release and impaired uptake of neurotransmitters (such as glutamate), excessive neuronal activation, cellular swelling, intracellular entry of divalent cations, and release of nitric oxide and free radicals. These changes in cell physiology lead to both apoptotic and necrotic cell death, and set in motion the development of a gliotic scar (ANKARCRONA et al., 1995; BACK and SCHULER, 2004; BONFOCO et al., 1995).
5.3.4 Microgliosis and neuronophagia
Microgliosis represented the fourth most frequently observed lesion (5.15%) in all treated animals even in those without degenerative neuronal changes (experimental group no. 2-14) except experimental group no. 6. The cause of this response remains undetermined. There is no correlation with doses, application mode, recovery period and the occurrence of the damage to the nervous tissue. The lack of immunolabelling of these cells with the lectin BS1 may reflect, that these cells with discrete thin processes were “ramified microglia” in the earliest state of activation, which are not identified by lectins (TILLOTSON and WOOD, 1989). Cell death is thought to activate microglial proliferation in experimental models of excitotoxicity and ischemia (LIU et al., 2001; DIHNÉ et al., 2001). However, even in the absence of
neuronal loss after transient global ischemia, microglial cells were proliferated suggesting that microgliosis does not require overt brain injury (LIU et al., 2001). In addition to cell death signals, various factors, such as macrophage colony-stimulating factor (M-SCF) granulocyte-macrophage colony-stimulating factor (GM-CSF), corticotropin-releasing hormone, and thrombin, can stimulate microglial cell proliferation (KOGUCHI et al., 2003; MITRASINOVIC et al., 2003; SUO et al., 2002;
WANG et al., 2003). The release of these factors together with or without cell death signals may synergistically contribute to the microglial proliferation observed during the treatment period. Microglia attempts to protect neurons in affected areas as well as to eliminate the detritus of necrotic cells. Microgliosis is characterized by a complex set of events, including changes in microglial morphology, increased proliferation, migration to a site of damage, phagocytosis, antigen processing and presentation, up-regulation of numerous cell surface proteins and secretion of signaling molecules, and apoptosis. Cell damage and death trigger the presentation of cell injury signals, such as membrane exposure of phosphatidylserine, or the release of adenosine 5’-triphosphate (ATP) and sialic acid containing glycosphingolipids (e. g., gangliosides), which induce proinflammatory cytokine and reactive oxygen species (ROS) production in microglia (MIN et al., 2004;
RATHBONE et al., 1999). Additionally, hypoxia, modulators from other cells, including certain surface molecules, cytokines, chemokines, and proteases can activate and also regulate the function of microglia. Interestingly, adenosine 5’-triphosphate (ATP) is also a regulatory signal molecule released from astrocytes that can serve as a chemotactic, mitogenic, and apoptotic signal for microglia (HONDA et al., 2001; RATHBONE et al., 1999; VERDERIO and MATTEOLI, 2001).
Microglial cells, the resident macrophages of the CNS are the primary source of innate and adaptive immune responses within the brain. They are main players in mediating neuroinflammatory cascades by expressing and/or releasing a number of different cytokines, chemokines, and receptors. Their ability to become activated throughout the course of neuropathic stimuli, such as invading pathogens, cell death, and hypoxia, allows microglia to respond to, and, at times, contribute to neuropathology (AARUM et al., 2003; BATCHELOR et al., 2002; MARIN-TEVA et al., 2004). Microgliosis is not only considered as the most immediate and harmful reaction of glial cells in the pathogenesis of acute CNS damage, but also prolonged microgliosis is known to exacerbate continuing damage in various neurodegenerative
disorders (HIRT et al., 2000). Microglial activation and phagocytosis may facilitate additional neuronal cell death, but recent studies showing release of several anti-inflammatory and neuroprotective factors during phagocytosis suggest that not all signals of cellular damage induce proinflammatory reactivity and toxicity in microglia (DE SIMONE et al., 2004; SUZUKI et al., 2004). It has not been resolved whether up-regulation of phagocytic function in microglia is beneficial or harmful. The clearance of pathogens, necrotic debris, and apoptotic cells is likely to promote healthy brain function and recovery from minor insults. Indeed, it has been reported that 24 hours after transient exposure to β-amyloid peptides, microglia continues to exhibit enhanced phagocytosis of several other unrelated substrates (KOPEC and CARROLL, 1998).
Activated microglia also releases a variety of ROS and reactive nitrogen species resulting in oxidative stress and increased neuronal death (BROWN and BAL-PRICE, 2003; COLTON et al., 2004; GAO et al., 2003). Like cytokines, these substances are also observed at high levels in various neurodegenerative conditions (ANDERSEN, 2004). Inhibition of the inducible nitrogen oxide synthase (iNOS) after lipopolysaccharide administration into the substantia nigra can rescue dopaminergic neurons from cell death whereas microglia-secreted superoxide also contributes to degeneration of dopaminergic neurons (ARIMOTO and BING, 2003; GAO et al., 2003).
5.3.5 Apoptosis
In this study, the presence of apoptotic cells after artemether treatment was evaluated by the TUNEL assay, but there was no evidence of apoptotic bodies or immunolabelled (TUNEL) apoptotic neurons even in animals exposed intramuscularly to high doses of artemether which caused neuronal degeneration and necrosis.
During apoptosis of various cell types, chromatin is degraded into high and low molecular weight fragments (OBERHAMMER et al., 1993; WYLLIE, 1980; BROWN et al., 1993; ZHIVOTOVSKY et al., 1994a, 1994b). Furthermore, apoptosis can occur locally without damage to adjacent healthy cells. This is in contrast to necrotic cell death, which exhibits rapid cell swelling and subsequent rupture of the plasma membrane. Since necrosis and apoptosis are biochemically and structurally different, they were originally classified as two separate forms of cell death. The morphologic
and biochemical changes during apoptotic cell death are mediated by a family of intracellular cysteine proteases named caspases. Activation of caspases also occurs by cleavage of aspartate residues (KERMER et al., 2004). Artemisinin derivatives seem to induce rather neuronal necrosis accompanied by reactive microgliosis than apoptosis.