Effects of different schedules of oxaliplatin treatment on the peripheral nervous system of the rat.

The aim of this study was to determine the influence of oxaliplatin scheduling on the onset of peripheral neurotoxicity and ototoxicity in a rat model. Animals were treated with four different schedules of oxaliplatin using two cumulative doses (36 and 48 mg/kg intraperitoneally (i.p.)). The neuropathological examination evidenced dorsal root ganglia (DRG) nucleolar, nuclear and somatic size reduction with nucleolar segregation in the treated rats. Sensory nerve conduction velocity (SNCV) was reduced after oxaliplatin treatment, while the auditory pathway was unaffected. After treatment, platinum was detected in the kidney, DRG and sciatic nerve. After a 5-week follow-up period, recovery of the pathological changes in the DRG and sciatic nerves occurred, although platinum was still detectable in these tissues. The following conclusions may be drawn: the main targets of oxaliplatin neurotoxicity were the DRG; the shorter the interval between the injections, the higher the severity of peripheral neuropathy and this was also related to the cumulative oxaliplatin dose; the peripheral neurotoxicity tended to be reversible; ototoxicity was absent even with high cumulative doses of oxaliplatin.

An ultrastructural study of neuronal changes in dorsal root ganglia (DRG) of rats after chronic cisplatin administrations.

In humans, the main dose-limiting side-effect of cisplatin (CDDP) treatment is a peripheral sensory neuropathy secondary to dorsal root ganglion (DRG) neuron involvement. To investigate further for neuronal alterations responsible for CDDP neurotoxicity we undertook the present experimental ultrastructural study, based on observations of 3 different groups of rats (6 animals in each group). Group A rats were treated with 1 mg/kg weekly for 9 weeks; Group B with 2 mg/kg weekly for 9 weeks; and group C rats served as untreated controls. At the end of the experiment, rats were perfused with 3% glutaraldehyde and lumbar DRGs were prepared for electron microscopic observations. In CDDP-treated rats somatic, nuclear and, above all, nucleolar size was reduced. Ultrastructurally, the nucleolus was the most affected structure. Nucleolar alterations were quantified morphometrically. Less marked changes were seen in the nucleus and in the RER and Golgi apparatus of the cytoplasm. The number of lysosomes and lipofuscins was greatly increased in CDDP-treated rats. The ultrastructural alterations observed in CDDP rats suggest that CDDP may be neurotoxic due to a reduction in protein synthesis. This assumption would explain why cells such as neurons, which are non replicating, but which have a high rate of protein synthesis, may be the target of the neurotoxic action of CDDP. The lack of an efficient blood/nerve barrier in the DRG explains the involvement of this particular type of neuron.

Effects of warm ischemia on valve endothelium.

BACKGROUND: This study investigates the time-dependent resistance of the endothelium of porcine aortic and pulmonary valves to different periods of warm ischemia (WIT). METHODS: Twenty-five 9-month-old swine were divided after death into five groups of WIT (0, 6, 12, 24, and 36 hours). Aortic and pulmonary valves were removed and a total of 15 aortic and 15 pulmonary valve specimens were obtained for each WIT interval. Valves were then examined for (1) their viability rate by the trypan blue dye exclusion method at light microscopy (percent of viability compared with 0 hours of WIT); (2) ultrastructural signs of irreversible or reversible ischemic damage by transmission electron microscopy (cell disruption, dilation of endoplasmic reticulum, cytoplasmic edema, nuclear and mitochondrial changes); (3) endothelial function by pharmacologic evaluation of both the endothelial-releasing capacity of prostacyclin and the endothelial-dependent dynamic responses to relaxing (acetylcholine from 1 x 10(-10) mol/L to 1 x 10(-4) mol/L) in aortic and pulmonary valve segments precontracted with norepinephrine (1 x 10(-6) mol/L) and contracting (NG-monomethyl-L-arginine, 1 x 10(-4) mol/L) drugs. RESULTS: Our results showed an endothelial progressive time-dependent ischemic injury, which reached significance after 12 hours of exposure. Viability and functional data indicated that 6 hours of WIT only provoked slight endothelial damage (p > 0.05 respect to time 0 hours), with signs at transmission electron microscopy consistent with a reversible injury. At 12 hours of exposure, we observed a significant reduction (p < 0.05) with respect to time 0 of the viability rate of prostacyclin production and of the endothelium-dependent dynamic responses to acetylcholine and NG-monomethyl-L-arginine. These functional impairments, although significant, were not consistent, however, with a complete loss of viability. Transmission electron microscopic observations confirmed the appearance of signs of irreversible injury; nevertheless, some elements were found to be well preserved or presented reversible damage. After 24 hours of WIT, ultrastructural and functional data were consistent with a dramatic decrease compared with controls in endothelial viability and functions (p < 0.01). Finally, after 36 hours of WIT, there was a subtotal loss of viability, of functions (p < 0.001) and, at transmission electron microscopic observations, of the endothelial layer of the valves. CONCLUSIONS: Our data show that the endothelial cells are resistant to short periods of WIT (up to 6 hours), and suggest that these cells can endure longer exposures, up to 12 hours of warm ischemia. Periods of 24 and 36 hours of WIT provoke progressive irreversible damage.

Low-dose glutathione administration in the prevention of cisplatin-induced peripheral neuropathy in rats.

So far various drugs have been used in an attempt to prevent or reduce cisplatin (CDDP)-induced peripheral neuropathy. Of those tried reduced glutathione (GSH) is one of the most promising. Its effectiveness has already been demonstrated by means of morphological methods in CDDP-treated rats in which high doses of GSH (up to 1200 mg/kg) were given. In the current study neurophysiological and morphological methods were used to evaluate the effect of low doses (150-300 mg/kg) of GSH i.p. on the peripheral nervous system of the rat. Four groups of 8 female Wistar rats were treated as follows: (A) CDDP 2 mg/kg i.p. weekly for 9 courses; (B) same as (A) plus GSH 150 mg/kg i.p. weekly; (C) same as (A) plus GSH 300 mg/kg i.p. weekly; (D) same as (A) plus GSH 150 mg/kg i.p. on the day of DDP injection followed by 150 mg/kg/day over the next 2 days. Eleven age-matched untreated rats were used as controls. Sensory conduction velocity was recorded in the tail nerve and morphologic and morphometric examinations were performed on the dorsal root ganglia neurons (L4-L6) in each animal. The results demonstrated that the neurophysiological and pathological changes induced by CDDP administration were less severe in rats co-treated with GSH. No significant differences could be related to the 3 different regimens of GSH co-treatments. This experiment confirms that GSH is able to reduce the neurotoxicity of CDDP and that it is effective even at doses as low as those used in the present study.

Circulating nerve growth factor level changes during oxaliplatin treatment-induced neurotoxicity in the rat.

BACKGROUND: Oxaliplatin neurotoxicity represents a clinically-relevant problem and its etio-pathogenesis is still unknown. We explored the possible role of some neuronal growth factors ("neurotrophins") during the course of oxaliplatin sensory neuronopathy. MATERIALS AND METHODS: In our rat model two different doses of oxaliplatin were used (2 and 3 mg/kg i.v. twice weekly for 9 times). The neurotoxicity of the treatment was assessed with neurophysiological and pathological methods and serum neurotrophin levels were measured by ELISA. RESULTS: Both oxaliplatin-treated groups showed the neurophysiological and neuropathological changes which mimic the chronic effects of oxaliplatin administration in humans, e.g. reversible sensory impairment due to dorsal root ganglia neuron damage. These changes were associated with a significant and dose-dependent reduction only in the circulating level of nerve growth factor (NGF), which returned to normal values after neurophysiological and pathological recovery. CONCLUSION: This specific association between neurological impairment and NGF modulation indicates that NGF impairment has a role in the neurotoxicity of oxaliplatin.

Effect on the peripheral nervous system of systemically administered dimethylsulfoxide in the rat: a neurophysiological and pathological study.

The issue of dimethylsulfoxide (DMSO) neurotoxicity is an important one, given its wide use in experimental toxicology as a solvent for hydrophobic substances. We examined the effect of the intraperitoneal administration of different DMSO solutions (1.8-7. 2%) on the peripheral nervous system of Wistar rats treated for 10 consecutive days and followed-up for an additional 45 days. DMSO administration induced a dose-dependent reduction in nerve conduction velocity, with complete recovery occurring in the follow-up. No structural changes were found in the sciatic nerve at 1.8% and 3.6% DMSO concentrations, suggesting that the mechanism of action of DMSO involves a functional impairment (i.e. conduction block) similar to that already described for this substance in isolated systems. However, when DMSO was administered at the 7.2% concentration, evident structural changes were observed in the sciatic nerve, with myelin disruption and uncompacted myelin lamelle. The neurophysiological and pathological changes observed in our study are severe enough to merit careful consideration in the course of experimental studies involving DMSO as a solvent for drugs which are under evaluation for their potential neurotoxicity.

Sural nerve bioptic findings in essential cryoglobulinemic patients with and without peripheral neuropathy.

Peripheral neuropathy often occurs in cryoglobulinemia but the pathogenesis of the peripheral nerve involvement is not completely understood, so that the relation between the reported endoneural changes and neuropathy is not clear. In this study we compared the sural nerve biopsies of 6 cryoglobulinemic patients with or without signs of peripheral neuropathy and all affected by the essential mixed type II form (ECII) and, moreover, of 8 age-matched controls. We found that in all the patients with neuropathy, axonopathy occurred and it was invariably associated with endoneural vessel damage. Moreover, the fiber losses were patchily distributed within the nerve fascicles. On the contrary both nerve fibers and vessels were normal in the patients without clinical and neurophysiological evidence of neuropathy and in controls. Our results support the hypothesis that the endoneural damage observed during ECII is not simply coincidental, but is relevant in the pathogenesis of cryoglobulinemic neuropathy. They also favor the assumption that ischemic damage of the nerve fibers occurs during ECII.

Ultrastructural aspects of DRG satellite cell involvement in experimental cisplatin neuronopathy.

Different substances may induce neurological impairment, clinically expressed as peripheral neuropathies, due to damage of the neuronal bodies (neuronopathy) of sensory or motor neurons. Neuronopathies have generally been studied referring to neurons, although other cellular components may also be damaged. Cisplatin (CDDP) is known to be neurotoxic to the neurons of the dorsal root ganglia (DRG). The scarcity of information as to the possible involvement and role played by dorsal root ganglion (DRG) satellite cells in neuronopathies prompted this study using the chronic DRG neuronopathy induced by the repeated administration of CDDP in rats as a model. Eighteen female Wistar rats were treated according to 3 different schedules of CDDP administration (6 rats for each group). Six further animals were used as controls. At the end of the experiment the L4-L5-L6 dorsal root ganglia were examined at the light and electron microscope. Ag-NOR reaction was also examined in 4 further CDDP-treated rats and 4 controls. Pathological changes in satellite cells of animals treated with CDDP were remarkable in the nucleus where heterochromatin clumps were reduced or even completely absent. Morphometric analysis of the area occupied by heterochromatin indicated that this nuclear component decreased in an exposure-time dependent manner. Frequently, nucleolar-like structures became apparent in the nucleus of the rats treated with the higher doses of CDDP. Ag-NOR positive regions in the nuclei of treated rats were increased with respect to the controls. Cytoplasmic changes in DRG satellite cells of CDDP treated rats were limited, being characterized by an increased electron-density of the matrix. In treated rats deep invaginations between satellite cells and the neuronal surface were evident, leading to the formation of vacuoli. The interstitial connective space often showed edematous areas. Our observations demonstrate that in chronic cisplatin neuronopathy, DRG satellite cells are also involved in the pathological changes induced by drug exposure, and that these changes may be interpreted as being mainly reactive.

Blood-nerve barrier of endoneural vessels in experimentally-induced hypothyroidism in rats.

Hypothyroidism may cause peripheral nerve damage, even if the pathophysiology of these changes is still unclear. It has been suggested by some that an increased vascular permeability is involved in hypothyroidism, while others have suggested a "compressive" mechanism caused by mucinous material deposited in the endoneurium. We have studied histologically the endoneurium and evaluated endoneural-vessel permeability in sciatic nerve by means of the leakage of horseradish peroxidase (HRP) in pharmacologically-induced hypothyroidism in rats. We did not find any substantial differences between the hypothyroid group of animals and the controls with respect to endoneural-vessel permeability. In particular, no macromolecular deposits were present in the extracellular space of the endoneurium in either the treated or the control rats. We therefore believe that a "vascular" hypothesis is unlikely for nerve involvement during hypothyroidism, nor was the "compressive" hypothesis supported by our histological findings.

Effects of repeated administration of low doses of cisplatin on the rat nervous system.

Cisplatin is a very effective antineoplastic drug. To date its major toxic dose-limiting effect is peripheral neuropathy. Whereas the clinical and neurophysiological features of cisplatin-induced neuropathy are fairly well known, its pathogenesis is still unclear. We treated a group of Wistar rats with low doses of cisplatin for 70 days in order to evaluate the light-microscopic and ultrastructural changes induced by chronic cisplatin administration in the spinal cord, spinal ganglia and peripheral nerves. Although the most striking pathological alterations were observed in the spinal ganglia neurons, initial axonal neuropathy was also demonstrated, whereas the spinal cord neurons were completely normal. Our findings further support the hypotheses that spinal ganglion neurons are the primary target of cisplatin peripheral neurotoxicity and that peripheral nerve damage is secondary to this neuronopathy.

The cytoarchitecture of the adult human parabrachial nucleus: a Nissl and Golgi study.

The parabrachial nucleus (PBN) plays important roles in numerous autonomic functions and in pain modulation. In different animal species, three main regions of the PBN have been identified: the m-PB, the l-PB, and the Kolliker-Fuse nucleus (KF). The KF has not been identified in humans. The present study used Nissl and Golgi-Cox material and morphoquantitative methods to investigate the cytoarchitectural organization of the adult human PBN, paying particular attention to neuronal features endowed with functional significance, i. e. the arborization of the neurons. The PBN neuron population is made up of elements which are heterogeneous in size, shape and dendritic arborization, and grouped into two regions, the lateral and medial PBN (l- and m-PB). It has been suggested that some large sized neurons located in the ventral region of the m-PB might be the counterpart of the KF. In the m-PB the fusiform neurons are the most numerous cells; in the l-PB the multipolar neurons prevail, and are particularly numerous in the dorsal l-PB. Since the dendritic arborization is generally the main target of afferent projections to a neuron, it is possible that the l-PB, and in particular its dorsal region, might be the main site for the endings of afferences to the human PBN.

Morphometric study of the sensory neuron and peripheral nerve changes induced by chronic cisplatin (DDP) administration in rats.

We performed a morphological, morphometric and toxicological study on the spinal ganglia and peripheral nerves of the rat after chronic administration of cisplatin (cis-dichlorodiammineplatinum II; DDP) with two different schedules. Severe damage of the spinal ganglia neurons was demonstrated with predominant involvement of the nucleus and nucleolus associated with a decrease in the cell size. Morphological and morphometric changes also occurred in the sciatic and peroneal nerves with the features of axonopathy. All these changes were more marked in the group of rats which underwent the most intense DDP treatment and the tissue platinum concentrations were also higher in this group. This experimental model is the first available for chronic DDP administration in which concomitant spinal ganglia and peripheral nerve damage has been confirmed pathologically. Our study supports the hypothesis that DDP-induced peripheral nerve fiber degeneration may result from nuclear and nucleolar changes in the sensory ganglion cell perikaryon.

A clinico-pathological and follow up study of 10 cases of essential type II cryoglobulinaemic neuropathy.

Ten patients with essential cryoglobulinaemia type II were examined for peripheral nerve damage. In six cases distal symmetrical nerve involvement was present, while in three other cases abnormalities restricted to single nerves were found. Electrophysiological and morphological data were consistent with axonal damage, the larger myelinated fibres being most affected. Although active signs of vasculitis and immunoperoxidase staining for immunoglobulins were not present, endoneurial vessels were widely damaged, with abnormally thick endothelial cells and redundant basal membranes. These findings, together with a patchy distribution of myelinated fibre loss, suggest ischaemia as a cause of peripheral neuropathy during essential cryoglobulinaemia type II. A follow up examination, performed one year after haematologial remission, revealed that no further peripheral nerve damage had occurred.

Ultrastructural and confocal laser scanning microscopical aspects of apoptosis in cultured human neuroblastoma SH-SY5Y cells.

Programmed cell death or apoptosis is a physiological process that plays an important role during development and maintains tissue homeostasis during adult life. In pathological conditions, such as cancer, apoptosis may be the mechanism by which cancer proliferation is hampered. Many antineoplastic drugs act by inducing apoptosis. SH-SY5Y human neuroblastoma cells undergo apoptosis when exposed to cisplatin, an effective antineoplastic drug. The occurrence of cell death by the apoptotic process is evidenced by the typical electrophoretic laddering of DNA, which begins 24 h after cisplatin exposure and becomes even more apparent at 3-4 days after exposure. Concomitantly, ultrastructural changes of the nucleus and nucleolar organization occur, followed by nuclear membrane disruption and, finally, by cytoplasm degeneration. These last two aspects are present in cultured cells detached from the substrate and predominate in long-term cultures after drug exposure. Confocal laser scanning microscopy (CLSM) of orange-acridine stained nuclei also clearly demonstrates the fragmentation of the chromatin into 3-5 domains. The CLSM, therefore can clearly demonstrate the occurrence of apoptosis in a much simpler, but equally accurate way than electron microscopy.

Re-innervation pattern of heterotopically transposed lingual flaps.

Pedicled tongue flaps have proved to be an effective method of repairing defects due to tissue loss in the oral cavity. No histological investigations have been done in respect of the longterm fate of these flaps after section of their nutrient pedicle. The histological pattern of the re-innervation process of heterotopically transposed lingual flaps in the oral cavity is evaluated in this paper. Two cases are reported: in the first, the tongue flap was used to repair the vermilion of the lower lip and in the second, for the closure of a post-traumatic defect of the hard palate. The histological findings are similar in the two cases: myelinated and unmyelinated fibres, free nerve endings and encapsulated receptors are present.

Endoneural vessel involvement in hypothyroidism.

Light-microscope findings and pathological ultrastructural changes in sural nerve biopsies from two patients affected by hypothyroidism, one with overt signs of peripheral neuropathy, the other asymptomatic, were studied. In both patients the endoneural vessels showed clear pathological changes similar to those of other metabolic neuropathies, but more marked in the symptomatic patient. It is proposed that the changes observed in the nerve fibers in hypothyroid neuropathy are secondary to changes in the endothelial cells and in the vessel wall.

Cryoglobulinaemic neuropathy: lack of progression in patients with good haematological control.

Nine consecutive patients with essential mixed cryoglobulinaemia type II (EMCII) were investigated by a follow-up study of 3-6 years (mean 5.3), to verify the long-term outcome of the peripheral nerve involvement in the disease. At the beginning of the study, a peripheral neuropathy, mild or moderate, mainly sensory, was present approximately in 50% of cases. All patients maintained good general conditions, low-staging of EMCII, were well controlled by therapy or did not need any medical treatment. During the neurological follow-up all patients had regular controls, usually at 1-year intervals, and only slight changes were seen with respect to the first evaluation. Therefore, we conclude that peripheral neuropathy during EMCII is less progressive that it can be thought at least in patients with benign course of the disease.