Carboplatin toxic effects on the peripheral nervous system of the rat.

BACKGROUND: The most striking of carboplatin's advantages (CBDCA) over cisplatin (CDDP) is its markedly reduced rate of neurotoxic effects. However, the use of CBDCA higher-intensity schedules and the association with other neurotoxic drugs in polychemotherapy may cause some concern about its safety with respect to peripheral nervous system damage. MATERIALS AND METHODS: Two different schedules of CBDCA administration (10 mg/kg and 15 mg/kg i.p. twice a week for nine times) were evaluated in Wistar rats. Neurotoxicity was assessed for behavioral (tail-flick test), neurophysiological (nerve conduction velocity in the tail nerve), morphological, morphometrical and analytical effects. RESULTS: CBDCA administration induced dose-dependent peripheral neurotoxicity. Pain perception and nerve conduction velocity in the tail were significantly impaired, particularly after the high-dose treatment. The dorsal root ganglia sensory neurons and, to a lesser extent, satellite cells showed the same changes as those induced by CDDP, mainly affecting the nucleus and nucleolus of ganglionic sensory neurons. Moreover, significant amounts of platinum were detected in the dorsal root ganglia and kidney after CBDCA treatment. CONCLUSIONS: CBDCA is neurotoxic in our model, and the type of pathological changes it induces are so closely similar to those caused by CDDP that it is probable that neurotoxicity is induced in the two drugs by the same mechanism. This model can be used alone or in combination with other drugs to explore the effect of CBDCA on the peripheral nervous system.

Experimental cisplatin neuronopathy in rats and the effect of retinoic acid administration.

Peripheral nervous system alterations were induced in adult rats by administration of cisplatin (CDDP) 2 mg/kg twice weekly for 4.5 weeks. Dorsal root ganglion neurons showed pathological changes. Morphometric determinations demonstrated a reduction in size of the somatic, nuclear and nucleolar area. The nucleoli were the most involved subcellular structures. Nerve conduction velocity and the tail-flick test for pain were both significantly altered in CDDP treated rats, whereas the rota-rod test for coordination revealed no changes in either treated or control rats. Analytical determinations demonstrated platinum accumulation in the DRG of CDDP treated rats. Spontaneous recovery, demonstrated by morphometric, electrophysiological, functional and analytical determinations, occurred after treatment discontinuation within about 7 weeks. A pilot study of the possible neuroprotective action of retinoic acid (RA) was also performed with this model of cisplatin neuronopathy. The rationale for using RA was based on its assumed antioxidant and neurotrophic effect. However, RA failed to prevent morphometric, electrophysiological, functional and analytical alterations induced by CDDP on DRG neurons. RA induced only a mild generalized protective effect.

The effects of irradiation at different times of the day on rat intestinal goblet cells.

Quantitative changes in jejunal goblet cells were studied in control and whole body irradiated rats using PAS-Alcian blue staining of crypt sections. A circadian dependence was observed when control animals were killed at different times during the light/dark cycle. Irradiation with 3 Gy produced a 2-3-fold increase within 36 h in goblet cells relative to controls, followed by a reduction to very low levels. There was a return to pre-treatment levels later than was observed for the columnar cells. The present results on the pattern of response of goblet cells and those of brush border enzyme activity are consistent with the hypothesis that ionizing radiation can influence differentiation. In fact during the first hours after irradiation an early induction of differentiation is evident while during the early repopulation phase columnar cells prevailed relative to the goblet cells. Only at later times were normal differentiation patterns seen. Groups of animals exposed to the same dose of radiation at different times of the day showed similar general patterns of behaviour even if the group irradiated at midnight showed a more marked and longer lasting injury.

Cell kinetics in rat small intestine after exposure to 3 Gy of gamma-rays at different times of the day.

Qualitative and quantitative morphological changes in rat jejunum were studied after a whole-body exposure to 3 Gy of gamma rays. Four groups of animals were irradiated at different times of the day, namely midnight, 06.00, 12.00 and 18.00 hours. The number of epithelial cells, labelling and mitotic indices were evaluated in crypt sections and the spatial distribution of S-phase cells was determined. At 12 h after irradiation a marked reduction was observed in all parameters, but the proliferative activity was restored quickly and at 36 h after irradiation the values were significantly higher than the controls. The frequency distribution of labelled cells at different positions in the crypt was reduced at 12 h but a clear expansion of S phase cells to positions near to the crypt villus junction was observed during the recovery phase. The animals irradiated at different times of the day showed a similar general post-irradiation response in the number of cells along the side of the crypt, labelling and mitotic indices and in the distribution of S phase cells along the crypts. It is worth noting that the animals exposed at midnight had a distribution of S phase cells similar to controls at 72 h post-irradiation, i.e. earlier than the other groups.

Off-treatment course of cisplatin-induced dorsal root ganglia neuronopathy in rats.

As yet little is known about the off-treatment course of cisplatin (CDDP) neurotoxicity. In this study we evaluated in the rat how the pathological changes in dorsal root ganglia (DRG) neurons and the electrophysiological alterations in peripheral nerves induced by chronic CDDP administration evolved after drug withdrawal. Twelve female Wistar rats were treated with CDDP and 3 of them were sacrificed 1, 5, 10 and 20 weeks after treatment. The results of the experiment indicated that: 1) the damage induced by sub-lethal doses of CDDP in the DRG neurons is reversible, 2) the first structures to be damaged are the DRG neurons (mostly their nucleoli) and 3) both damage and recovery in the follow-up period involve first the DRG neurons and subsequently the peripheral nerves.

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.

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.

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.

Cell proliferation and differentiation in the small intestine after irradiation with multiple fractions.

Qualitative and quantitative morphologic changes in rat small intestine were studied after abdominal exposure to multiple fractions of gamma radiation. One group of animals received 3 X 2 Gy with one fraction every 4 hours. Another group received two courses of this type with a 16 hour interval between the courses (total dose 6 X 2 Gy). A marked decrease in the number of crypt epithelial cells, and in mitotic and labelling indices, was observed up to 24 to 36 hours after the end of both regimens. Repair and recovery occurred within 72 hours after the end of the last exposure, and the epithelium regained normal morphology. At 1 and 4 hours after the end of the treatment the frequency of S-phase cells along the crypt was greatly reduced and at the following intervals labelled cells occupied the region where differentiation occurs in control animals. During recovery labelled cell distribution showed a gradual return to normal. No substantial differences between the effects of total doses of 6 and 12 Gy were shown except for a greater reduction in crypt epithelial cells at the early time intervals after the larger dose.

Quantitative changes in the goblet cells of the rat small intestine after irradiation.

In order to evaluate the process of cell differentiation in the crypt of the rat small intestine the goblet cells were quantitatively studied in controls and after irradiation of the abdomen. The effect of a single dose, administered at 4 different times of the day, and multiple daily fractionations (MDF) of 6 and 12 Gy with different doses per fraction and different time intervals, were compared. Both regimens caused an initial increase of the goblet cells (both in absolute and relative terms), followed by a decrease and finally return to nearly control levels. After MDF the increase was more marked and the return to a normal level occurred earlier than after the single dose.

Qualitative and quantitative effects on the morphology of the small intestine after multiple daily fractionation.

The behaviour of qualitative and quantitative morphologic parameters in rats exposed on the abdomen only to multiple daily fractionation (MDF) of 3 Gy X 2 and 3 Gy X 2 X 2 with 12 h splits was analyzed. Both doses were sublethal. The number of epithelial cells in the crypts and in the whole formation, the mitotic index, and the labelling index appeared to be markedly reduced at early intervals after irradiation. Repair and recovery quickly led to normal morphology. The effects of an 8 Gy single dose were compared with those from multiple fractionations.

Modifications of S-phase cell distribution in the intestinal crypts after multiple daily fractionation.

The effects obtained by multiple daily fractionation (3 Gy X 2 or 3 Gy X 2 X 2) on the distribution of S-phase cells along the crypt of the small intestine were investigated. The frequency of labelled cell distribution was reduced at early intervals; then the proliferating compartment gradually extended to the villus junctions. During recovery labelled cell frequency in the lower half of the crypts returned to control levels, while labelled cells were present in the differentiating area. With lower total dose modifications were milder and, as early as 72 h before exposure, distribution was already similar to controls. Invertase activity showed an initial increase and a higher reduction during acute damage when fractionation with higher doses was used. A lack of return to normal activity was present even 11 days after exposure when, however, the characteristic circadian pattern was observed.

S-phase cell distribution in the small intestine irradiated at different times of the day. I. Acute irradiation injury.

The S-phase cell distribution has been analysed to evaluate the behaviour of proliferative cells in the intestinal epithelium after irradiation at different times of the day. A marked reduction of S cell frequency was observed at early intervals after abdominal irradiation; this reduction was particularly evident in the lower half of the crypts. At subsequent intervals a progressive extension of the proliferative compartment, with labelled cells also at the top of the crypt, was present. The irradiated groups generally showed a homogeneous behaviour even if a more marked reduction in S-phase cells was observed in group C. The invertase activity, a brush border enzyme synthetized during the differentiation process, presented a different behaviour at the early intervals in the irradiated groups. When the extension of the proliferative compartment occurred the invertase activity reached values close to zero. The modifications in brush border enzymes and in S-phase cell distribution, at early killing times, led to the hypothesis of an early differentiation.

S-phase cell distribution in the small intestine irradiated at different times of the day. II. Recovery phase.

Modifications occurring during recovery in the small intestine of animals exposed to the same radiation dose given at different times of the day were evaluated. S-phase cell distribution along the crypts and invertase activity were evaluated to ascertain the functional capacity of epithelial cells. In animals killed between 5 and 6 days after exposure, S-phase cell distribution and functional conditions tended towards normality although recovery was not complete. Labelled cells occurred also at villus junctions, demonstrating limitation in size of the differentiating compartment. This was confirmed by reduced activity of the brush border enzymes. Animals irradiated at the end of the dark period recovered more quickly and efficiently. In this group, labelled cell distribution was almost the same as in the controls starting from 120 h, and invertase activity was also closer to the controls than in any other group.

Behaviour of the proliferative compartment of the small intestine at different times of the day.

The modifications of mitotic and labelling indices in animals killed at different hours of the day were analysed. The invertase activity was also assayed. This brush border enzyme, synthetized by the differentiating cells, showed a clear circadian rhythm with a maximum in the night and a minimum near the end of the light period. Mitotic and labelling indices showed the highest activity in the night and at noon. The positions of mitotic and labelled epithelial cells in the crypt were also determined. The frequency of labelled cells in the different positions of the crypt evidenced a shift of these cells towards the crypt-villus junction in the late afternoon with a consequent reduction of the differentiating compartment.

Irradiation at different times of the day. Morphology and kinetics of the small intestine.

Rats were irradiated at different time of the day with sublethal doses on the abdomen only, and qualitative and quantitative morphologic modifications were determined. The experiments seemed to be demonstrate that in the groups irradiated at night and at the end of the light period early injury is not severe whereas in the group irradiated at the end of the dark period repair of the injury seems to be more effective.