- Email: [email protected]
Changes in cholinergic responses of sweat glands during denervation and reinnervation
Journal of the Autonomic Nervous System 74 Ž1998. 134–142
Changes in cholinergic responses of sweat glands during denervation and reinnervation Jorge J. Vilches, Francisco J. Rodrıguez, Enrique Verdu, ´ ´ Antoni Valero, Xavier Navarro
)
Department of Cell Biology and Physiology, Unitat de Fisiologia, Faculty of Medicine, UniÕersitat Autonoma de Barcelona, E-08193 Bellaterra, ` Barcelona, Spain Received 4 May 1998; revised 3 August 1998; accepted 9 September 1998
Abstract Functional sudomotor responses have been studied in sweat glands reinnervated after sciatic nerve crush and partially denervated by cisplatin intoxication in the mouse. The sudomotor function mediated by the sciatic nerve was evaluated by silicone imprints on the plantar surface of the hindpaws. Five days after nerve crush, completely denervated sweat glands became unresponsive to cholinergic stimulation with pilocarpine. During the following weeks, the number of reinnervated, reactive sweat glands increased progressively to reach a maximum of 89% of preoperative control counts by 40 days after nerve crush. At this time, the mean volume of sweat secreted per gland was normal, but reinnervated glands showed a secretory activity abnormally sustained over time after pilocarpine stimulation and, on the other hand, had an increased resistance to the inhibition of secretion induced by atropine. The effects of cisplatin administration on sudomotor function were investigated in two groups of mice, one treated with high doses of cisplatin Ž10 mgrkgrweek for 4 weeks. and another treated with low doses of cisplatin Ž5 mgrkgrweek for 8 weeks.. Cisplatin intoxication produced abnormal sudomotor responses indicative of denervation from cumulative doses of 10 mgrkg. The first abnormality found was a partial resistance of sweat glands to atropine, followed by a decrease in the sweat output per gland and finally a decline in the number of sweat glands activated by pilocarpine. These abnormalities in the sudomotor responses were more pronounced in mice treated with a high dose than in those with a lower dose regime. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Sweat glands; Cholinergic stimulation; Autonomic neuropathy; Denervation; Nerve lesion; Cisplatin
1. Introduction Sweat glands ŽSGs. are innervated by sympathetic postganglionic axons, that in contrast to the ordinary sympathetic innervation, use acetylcholine as the principal neurotransmitter ŽDale and Feldberg, 1934; Dobson and Sato, 1972; Stevens and Landis, 1987.. The cutaneous location of the SGs makes it possible to evaluate the functional activity of the sympathetic division of the autonomic nervous system with noninvasive methods such as colorimetric, evaporimetric and imprint techniques ŽMinor, 1927; Low et al., 1983; Kennedy et al., 1984a,b; Kennedy and Navarro, 1993.. Experimental evaluation of sudomotor function in intact animals has become possible by silastic imprints ŽKennedy and Sakuta, 1984; Kennedy and Navarro, 1993.. This method allows an accurate quantita) Corresponding author. Tel.: q34-9358-11966; fax: q34-9358-12986; e-mail: [email protected]
tion of the SG secretion, repeatedly over time, in order to follow changes of sudomotor function that occur either during natural processes such as maturation and aging ŽNavarro et al., 1988; Verdu´ et al., 1996., or after different types of nerve lesions ŽKennedy and Sakuta, 1984; Bharali et al., 1988; Navarro and Kennedy, 1989; Cardone and Dyck, 1990; Verdu´ et al., 1995.. SGs are apparent exceptions to the law of denervation hypersensitivity proposed by Cannon Ž1939.. Following an acute nerve lesion, denervated SGs become unresponsive to cholinergic agonists ŽList and Peet, 1938; MacMillan and Spalding, 1969; Kennedy and Sakuta, 1984; Navarro et al., 1988.. In previous reports we showed that activation of secretion of denervated SGs by pilocarpine was completely absent within 1 week after crushing or freezing the sciatic nerve of rodents. Reactive SGs first reappeared by 18–23 days after such nerve injuries and increased progressively in number to reach a maximum of approximately 90% of the pre-lesion baseline values at day 40,
0165-1838r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 1 8 3 8 Ž 9 8 . 0 0 1 5 2 - 0
J.J. Vilches et al.r Journal of the Autonomic NerÕous System 74 (1998) 134–142
135
Fig. 1. Silicone molds of the plantar surface of a mouse showing the sweat impressions obtained Ža. in a baseline preoperative test and Žb. 7 days after sciatic nerve crush and saphenectomy.
then remained about the same 2 months later ŽNavarro et al., 1988; Navarro et al., 1994; Verdu´ et al., 1995; Verdu´ and Navarro, 1997.. The progress of sudomotor nerve regeneration was judged by the reappearance of secreting SGs over time, thus, concluding that functional reinnervation was completed in the mouse by 40 days after nerve lesion. However, partially denervated SGs remain sensitive to pilocarpine ŽKennedy and Sakuta, 1984; Kennedy et al., 1984a,b.. This means that silastic imprints of SG secretion induced by cholinergic agents do not detect intermediate stages of sudomotor denervation. If more about the sudomotor responses to cholinergic agonists and antagonists was known, then partial innervation of SGs might become detectable using these techniques. Cisplatin Ž cis-dichlorodiammineplatinum II, DDP. is an antineoplastic drug widely used in the treatment of many common tumors including those affecting ovary, testes and
bladder. The compound has several toxic side-effects, but at present, neurotoxicity is considered to be the major dose-limiting effect. DDP causes a predominantly sensory peripheral neuropathy ŽRoelofs et al., 1984; Thompson et al., 1984; Boogerd et al., 1990. with occasional autonomic dysfunctions ŽRosenfeld and Broder, 1984; Boogerd et al., 1990.. Cisplatin administration to rats under different dose regimes induces early morphological changes in dorsal root ganglia neurons ŽTomiwa et al., 1986; Cavaletti et al., 1992., supporting the hypothesis that DDP induces a primary neuronopathy of dorsal root ganglia neurons followed by a mild axonopathy mainly involving large myelinated fibers ŽCavaletti et al., 1992; Gao et al., 1995; Barajon et al., 1996.. Similar pathological changes have been described in ganglionic sympathetic neurons ŽTredici et al., 1993., that may underlie the autonomic disfunctions reported following DDP therapy. The aim of the present study was to evaluate the functional changes of cholinergic sudomotor responses in Table 1 Comparison of the total number of reactive sweat glands ŽNo SGs. and the sweat output per gland ŽSOrGS. 10 min after pilocarpine injection, and percentage of SGs reactive to pilocarpine 20 min after atropine administration Ž% SGs. in the operated hindpaw 40 days after a sciatic nerve crush ŽCX. and in the contralateral intact hindpaw ŽCTL. from the same mice Paw
Parameter No SGs
CX CTL Fig. 2. Total number of reactive sweat glands ŽNo SGs. in the hindpaw 10 min after pilocarpine injection during the reinnervation process after sciatic nerve crush. Values are expressed as means and S.E.M.
296.6"12.5 365.5"18.5
a
SOrSG Žnl.
% SGs
0.22"0.02 0.21"0.01
91.6"3.3 a 49.4"9.7
Values are expressed as means"S.E.M. a p- 0.05 vs. CTL.
136
J.J. Vilches et al.r Journal of the Autonomic NerÕous System 74 (1998) 134–142
denervation and reinnervation processes. In order to assess the time course and severity of functional abnormalities, we made serial testing after either an acute nerve lesion by crushing the sciatic nerve or different cumulative doses of DDP. 2. Materials and methods 2.1. Experimental design Four groups of Swiss female mice, aged 2.5 months at the beginning of the experiments, were used. Animals of
Fig. 4. Total number of reactive sweat glands ŽNo SGs. after pilocarpine injection in groups HD Žtop., LD Žmiddle. and CTL Žbottom.. Cumulative doses of DDP in the legend are expressed as mgrkg. Values are shown as means and S.E.M.
Fig. 3. ŽA. Total number of reactive sweat glands ŽNo SGs. in the hindpaw and ŽB. sweat output per gland ŽSOrSG. after pilocarpine injection 40 days after sciatic nerve crush, in the operated hindpaw ŽCX. and in the contralateral intact hindpaw ŽCTL. from the same mice. Values are shown as means and S.E.M.; time is in minutes after pilocarpine injection. ŽC. Effects of atropine on the glandular stimulation induced by pilocarpine 43 days after sciatic nerve crush with doses of atropine 0.05 mgrkg ŽCX 0.05. or 0.1 mgrkg ŽCX 0.1. and 0.05 mgrkg in the intact hindpaw ŽCTL 0.05. from the same mice. Values are expressed as means and S.E.M. of the percentage of reactive sweat glands Ž% SGs. with respect to the total number responsive to pilocarpine. Time is in minutes after atropine administration.
one group Ž n s 10. were subjected to a sciatic nerve crush. Under pentobarbital anesthesia Ž50 mgrkg, i.p.., the saphenous nerve was cut in the femoral space and a long segment of the distal stump removed to prevent regeneration. The sciatic nerve was then exposed and crushed three times in succession with a fine forceps ŽDumont no. 5. at 45 mm from the tip of the third digit. Finally, the skin was closed with 4-0 sutures and disinfected with povidone iodine solution. The number of reactive SGs 10 min after pilocarpine injection Ž5 mgrkg, s.c.. was evaluated before operation and at several intervals up to 90 days post-operation. Since by 40 days after sciatic crush the number of SGs reactive to pilocarpine achieves maximal values, similar to pre-operative controls ŽNavarro and Kennedy, 1989; Verdu´ et al., 1995., we made specific tests in order to detect changes in the cholinergic responses by 40 and 60 days. As the number of SGs on right and left paws are essentially equal ŽNavarro et al., 1988., these tests were also done in the contralateral paw as control.
J.J. Vilches et al.r Journal of the Autonomic NerÕous System 74 (1998) 134–142
In the second experimental paradigm three groups of mice were used. One group ŽHD, n s 10. was injected with DDP ŽNeoplatin w , Bristol-Myers Squibb. dissolved in sterile saline solution, 10 mgrkg i.p. once a week for 4 weeks. This group was evaluated before the onset of treatment and biweekly at cumulative doses of 20 and 40 mgrkg. A second group ŽLD, n s 9. was given DDP at a lower dose, 5 mgrkg i.p. once a week for 8 weeks. This group was evaluated before the onset of DDP treatment and biweekly at cumulative doses of 10, 20, 30 and 40 mgrkg. To prevent DDP-induced renal damage, 1 ml of saline solution was injected subcutaneously immediately after DDP administration. In a control group ŽCTL, n s 8. animals were treated with saline intraperitoneally once a week for 8 weeks. 2.2. Functional tests The number of secreting SGs and the sweat output per gland was determined by making an impression mold of the plantar surface of the hindpaw using a silicone material ŽElasticon, Kerr, Romulus, MI.. A mixture of 0.2 ml base material plus four drops of hardener from a 23-gauge needle was gently spread with a spatula over the sole. As the silicone hardened Žduring approximately 3 min., it retained the impressions of the sweat droplets as they emerged from the sweat ducts and pushed up into the mold. Each indentation has been shown by histological methods to mark the location of one sweat duct ŽKennedy et al., 1984a,b.. Droplet impressions were counted under a dissecting microscope Ž=20. using transmitted light. Total counts of SGs were made for each paw and for defined subdivisions of the paw ŽKennedy and Sakuta, 1984; Kennedy et al., 1984a,b.. The diameter of each sweat impression in the third toe flat area was measured using a micrometer grid, and used to calculate the volume of sweat
137
droplets considered to approximate a sphere Žvolumes 4r3 p r 3 . ŽKennedy et al., 1984a,.. For the assessment of sudomotor responses to cholinergic stimulation, pilocarpine nitrate Ž5 mgrkg. was injected subcutaneously into the back of the mice, as this dose of pilocarpine produces a maximal and stable sudomotor response in the mouse ŽVilches et al., 1995.. Silicone molds were made 5, 10, 15, 20, 30 and 45 min after injection. For evaluating the antagonistic inhibition of sweating, a dose of 0.05 mgrkg of atropine was injected subcutaneously 15 min after pilocarpine injection. Silicone molds were made 10 min after pilocarpine injection Žmaximal number of reactive SGs. and 5, 10, 15 and 20 min after atropine administration. In order to abolish emotional or basal sweating, the distal innervation of the paw was blocked by injecting 0.05 ml of bupivacaine Ž0.75%. at the ankle before the pilocarpine injection ŽVilches et al., 1995.. Values of sudomotor responses to pilocarpine stimulation are shown as the absolute number of sweat impressions in the mold and the estimated sweat output per SG, whereas results obtained after atropine administration are expressed as the percentage of the number of impressions found 10 min after pilocarpine injection. All results are shown as means and S.E.M. Statistical comparisons between values obtained from different groups of mice were made by ANOVA with the Scheffe´ test for multiple comparisons, and comparisons between values obtained from the same mice by the paired t-test.
3. Results 3.1. Sudomotor responses after nerÕe crush After sciatic nerve crush, the number of plantar SGs activated by pilocarpine declined daily and reached zero
Table 2 Results of the total number of reactive SGs ŽNo SGs. and the sweat output per gland ŽSOrSG. 10 min after pilocarpine injection, and percentage of SGs reactive to pilocarpine Ž% SGs. 20 min after atropine administration in control mice ŽCTL. and mice treated with low ŽLD. and high ŽHD. doses of DDP Parameter
No SGs
SOrSG Žnl.
% SGs
Group
CTL LD HD CTL LD HD CTL LD HD
Cumulative dose Žmgrkg. Baseline
10
20
30
40
400.5 " 11.0 404.8 " 10.5 420.3 " 10.5 0.15 " 0.02 0.16 " 0.02 0.14 " 0.02 23.8 " 11.2 7.0 " 1.9 25.8 " 14.2
413.0 " 12.6 415.3 " 14.3
415.6 " 10.7 405.3 " 14.8 358.7 " 25.2 0.17 " 0.02 0.16 " 0.02 0.13 " 0.02 37.2 " 11.7 46.3 " 9.8 c 86.9 " 6.0 a,b,c
411.9 " 8.4 389.7 " 15.8
406.3 " 9.0 306.4 " 37.0 a,c 148.1 " 44.8 a,b,c 0.17 " 0.03 0.08 " 0.01a,c 0.03 " 0.01a,b,c 20.9 " 8.0 69.8 " 10.6 a,c 91.3 " 5.1 a
0.17 " 0.02 0.19 " 0.01 39.6 " 7.7 53.3 " 10.8 c
0.16 " 0.02 0.11 " 0.01a,c 41.7 " 9.6 77.7 " 4.9 a,c
Values are expressed as means" S.E.M. n s 8, 9 and 10 for groups CTL, LD and HD respectively, except for the test of atropine inhibition at a cumulative dose of 40 mgrkg, at which the number of mice was 8, 6 and 4 for groups CTL, LD and HD, respectively. a p - 0.05 vs. CTL. b p - 0.05 vs. LD. c p - 0.05 vs. baseline values obtained before the onset of treatment.
138
J.J. Vilches et al.r Journal of the Autonomic NerÕous System 74 (1998) 134–142
response in reinnervated SGs ŽTable 1.. However, a higher dose of 0.1 mgrkg atropine produced a significant reduction of the percentage of secreting SGs in the operated paw ŽFig. 3C.. Similar results were obtained at postoperative days 60 and 63, in the SG responses to pilocarpine as well as in the inhibition induced by atropine. 3.2. Sudomotor responses during DDP intoxication Fig. 4 shows the number of SGs responsive to pilocarpine in HD, LD and CTL groups. The highest number of reactive SGs was observed 10 min after pilocarpine administration in the three groups of mice. The number of reactive SGs decreased progressively with increasing cumulative doses of DDP in treated groups, more markedly in group HD than in group LD, although the maximal number of reactive SGs was significantly lower with re-
Fig. 5. Sweating response in two footpads of a mouse from the LD group Ža. before the onset of DDP treatment and Žb. at a 40 mgrkg cumulative dose.
by day 5 ŽFig. 1.. The first reactive SGs appeared two weeks after lesion and their number increased rapidly to reach a maximum of 89% of the preoperative values at 40 days postoperation. This number did not increase at 60 and 90 days tests ŽFig. 2.. Despite the fact that 40 days after nerve lesion the maximum number of reactive SGs in the denervated paw was lower than in the intact contralateral paw of the same mice, this difference was only observed 10 min after pilocarpine injection ŽTable 1.. In the following molds the number of reactive SGs in the reinnervated paw did not differ significantly from those in the contralateral paw, due to the slight decline in the number of secreting SGs in the control paw in contrast to the stable response of the operated paw ŽFig. 3A.. On the contrary, 40 days after nerve crush the sweat output per gland was similar in both paws; furthermore, 45 min after pilocarpine injection the volume secreted by reinnervated SGs was slightly larger than that produced by control SGs ŽFig. 3B.. The reinnervated SGs showed a partial resistance to atropine 43 days after nerve crush. Whereas in the control paw a dose of 0.05 mgrkg of atropine produced a marked decline in the percentage of SGs reactive to pilocarpine, the same dose did not blocked the pilocarpine-induced
Fig. 6. Sweat output per gland ŽSOrSG. after pilocarpine injection in groups HD Žtop., LD Žmiddle. and CTL Žbottom.. Cumulative doses of DDP in the legend are expressed in mgrkg. Values are shown as means and S.E.M.
J.J. Vilches et al.r Journal of the Autonomic NerÕous System 74 (1998) 134–142
spect to controls only at a 40 mgrkg cumulative dose ŽTable 2, Fig. 5.. The number of reactive SGs to pilocarpine tended to be abnormally sustained from 10 to 45 min after injection in DDP-treated mice, while in control mice there was a decline most evident between 10 and 20 min molds. Similar findings were observed for the sweat output per gland ŽFig. 6.. The maximal volume secreted per reactive SGs was achieved 10 min after pilocarpine administration, both in control and DDP-treated mice. The sweat output per gland also decreased progressively with increasing cumulative doses, more in group HD than in group LD. The decrease in the sweat volume was more pronounced than the decline in number of secreting SGs, reaching a significant difference with respect to controls by cumulative doses of 30 mgrkg ŽTable 2..
Fig. 7. Effects of atropine on the SG stimulation induced by pilocarpine in groups HD Žtop., LD Žmiddle. and CTL Žbottom.. Cumulative doses of DDP in the legend are expressed in mgrkg. Values are shown as means and S.E.M. of the percentage of reactive sweat glands Ž% SGs. with respect to the number responsive to pilocarpine. Time is in minutes after atropine administration.
139
Atropine produced a strong reduction in the percentage of SGs reactive to pilocarpine in group CTL as well as in the baseline results of groups HD and LD mice. However, DDP intoxication caused an increased resistance of reactive SGs to atropine, that was more evident in group HD than in group LD ŽFig. 7.. The resistance to atropine was statistically significant from cumulative doses of 10 mgrkg ŽTable 2. and tended to increase at higher doses, while the results of the control group did not change notably throughout the follow-up, neither for pilocarpine-induced responses nor for atropine inhibition.
4. Discussion The insensitivity of denervated SGs to cholinergic agonists provides a convenient system to evaluate the degree of functional innervation of SGs by sudomotor sympathetic nerves in situations that may cause denervation and reinnervation processes ŽKennedy and Navarro, 1993.. By further investigating the sudomotor responses to cholinergic agonists and antagonists, we have found that SGs which are presumably only partially innervated, as a consequence of reinnervation after nerve injury, show abnormal responses, such as a more sustained secretion over time after stimulation and a partial resistance to the inhibitory effect of atropine. Denervation of SGs as a consequence of traumatic peripheral nerve lesions lead to their lack of reactivity to cholinergic stimuli. After complete axotomy by sectioning, crushing or freezing the mouse sciatic nerve the number of secreting SGs decreases exponentially to zero during the first week ŽKennedy and Sakuta, 1984; Navarro et al., 1988; Navarro and Kennedy, 1989., as also confirmed in the present study. Sudomotor regeneration, judged by the reappearance of pilocarpine-reactive SGs, was progressive and reached the maximum by 40 days after nerve crush, in agreement with the results of previous reports ŽNavarro and Kennedy, 1989; Navarro et al., 1994; Verdu´ et al., 1995; Verdu´ and Navarro, 1997.. Focal lesions produced by freezing or crushing peripheral nerves cause complete axotomy ŽBridge et al., 1994., but do not disrupt the basal lamina surrounding each nerve fiber or the epineural sheaths ŽHaftek and Thomas, 1968; Mira and Fardeau, 1978., thus allowing an efficient regeneration and an appropriate reinnervation of target organs. The number of reinnervated reactive SGs after a crush injury reaches close to normal values. Nevertheless, in our study newly reinnervated SGs showed peculiar abnormal cholinergic responses. A sustained secretory activity was observed in reinnervated SGs, manifested by the stability in the number of reactive SGs and in the sweat output per gland over time after pilocarpine administration. This evidence is compatible with the long duration of SG reactivity reported by Kennedy and Sakuta ŽKennedy and Sakuta, 1984. a few hours after sciatic nerve section in the mouse,
140
J.J. Vilches et al.r Journal of the Autonomic NerÕous System 74 (1998) 134–142
as well as with the overrepresentation of large diameter droplets reported in diabetic patients with mild neuropathy ŽKihara et al., 1993.. On the other hand, SGs of the lesioned paw were more resistant to the inhibition induced by atropine than SGs of the contralateral intact paw. Although a positive correlation between functional sudomotor recovery and the density of PGP 9.5-immunoreactive nerve fibers around the SGs has been found after sciatic nerve crush in the mouse, the density of sudomotor fibers even 100 days postoperation was about 50% lower than in unoperated control samples ŽVerdu´ and Navarro, 1997.. This finding supports the view that partially innervated SGs remain responsive to cholinergic stimulation. Thus, the abnormal cholinergic responses of reinnervated SGs showed in the present study may be a functional evidence of a deficit in SG innervation. The results of this study also show that chronic administration of DDP induces a decline in the number of reactive SGs and the sweat output per gland. Functional disturbances developed progressively with higher cumulative doses and were more pronounced in mice treated with a high dose than in those with a lower dose regime. These findings are compatible with the lack of responsiveness of denervated SGs in nerve lesions and in toxic or metabolic neuropathies. In diabetic subjects, several studies have shown a decrease in the number of reactive SGs, the sweat rate and the sweat output after cholinergic stimulation ŽLow et al., 1983; Kennedy et al., 1984a,b; Kennedy and Navarro, 1989; Stewart et al., 1994.. Similar results have been reported in alcoholic neuropathy ŽNavarro et al., 1993a,b. and in experimental acrylamide intoxication ŽNavarro et al., 1993a,b.. All these findings suggest that DDP intoxication affects sympathetic postganglionic function. Morphological studies of peripheral nerves of DDPtreated rodents showed degenerative signs and loss of myelinated nerve fibers to a mild degree, more evident in distal than proximal nerves, and little or no change in unmyelinated fibers ŽCavaletti et al., 1992; Gao et al., 1995.. However, the similar ultrastructural changes observed in dorsal root and sympathetic ganglia neurons ŽTomiwa et al., 1986; Cavaletti et al., 1992; Tredici et al., 1993; Barajon et al., 1996. support the hypotheses that these cells are the primary targets of DDP-induced neurotoxicity, due to the absence of an efficient blood–nerve barrier at these two sites. Moreover, DDP inhibits the neurite outgrowth in cultures of rat superior cervical ganglion ŽHayakawa et al., 1994.. It has been reported that DDP reduces the levels of secretory neuropeptides both in dorsal root ganglia ŽApfel et al., 1992; Schmidt et al., 1995. and in the distal nerve fibers innervating the skin and the SGs ŽVerdu´ et al., 1998.. The loss of neurotransmitter supply, due to an impairment of the axonal transport ŽRussell et al., 1995., might lead to an early decline in sudomotor function, followed by later structural denervation ŽVerdu´ et al., 1998.. Nevertheless, changes in sudomotor responses of DDP-treated mice might be also due to
a direct toxic effect of DDP on SGs. Thus, further studies are necessary to determine the toxicity of DDP on SGs and postganglionic sudomotor neurons. In our study, the decrease in the number of reactive SGs was significant when reaching a DDP cumulative dose of 40 mgrkg. SGs that still remained reactive to pilocarpine showed an abnormally maintained response, similar to that found in reinnervated SGs after nerve crush. An excessive, prolonged sweating response has also been described in some diabetic patients after cholinergic stimulation ŽLow et al., 1983; Levy et al., 1991.. The decrease in the sweat output per gland was more marked and appeared earlier than the decline in number of reactive SGs. The partially denervated SGs would produce smaller sweat droplets, before ceasing secretion when completely denervated. This evidence is in agreement with findings from a large series of diabetic patients, in whom about a 15% had a reduced mean volume of sweat secretion with a normal number of reactive SGs ŽKennedy and Navarro, 1989.. On the other hand, an increased resistance of SGs to atropine inhibition was observed already at cumulative doses of 10 mgrkg, indicating that it may be considered as the first sign of sudomotor impairment in DDP-treated mice. This sudomotor abnormality even precedes the appearance of sensory nerve conduction slowing ŽVerdu´ et al., 1998.. Therefore, these findings would recommend to perform a thorough evaluation of sudomotor activity in patients treated with DDP, in order to detect its neurotoxicity at early stages. Relatively little is known about the loss of responsiveness of denervated SGs. The insensitivity of denervated SGs could be due to a decline in number or function of SG muscarinic receptors. The effect of cholinergic denervation on muscarinic receptor expression is diverse in other peripheral targets. Denervation of cat iris produces no change in the concentration or affinity of muscarinic receptors ŽSachs et al., 1979.. In contrast, parasympathectomy of the rat urinary bladder provokes an increase ŽNilvebrant et al., 1986., whereas denervation of rat parotid gland causes a decrease in the density of muscarinic receptors ŽTalamo et al., 1979.. However, in all these targets denervation produces hypersensitivity to cholinergic agonists, suggesting that regulation of the functional responsiveness does not depend of the receptor expression. The case of denervated SGs supports this hypothesis, as the density, the level of expression and the distribution of SG muscarinic receptors do not change 7 days after transection of the rat sciatic nerve ŽGrant et al., 1991.. Furthermore, the molecular subtype of muscarinic receptor and its affinity were also unchanged after denervation. Another explanation of nonresponsiveness is that the functional receptor was present but uncoupled from the secretory pathway. Since G-protein coupling is unaffected by denervation ŽGrant and Landis, 1991., the most likely cause of SGs insensitivity would be a change in a step of the pathway distal to the G-protein. We noticed that the first responsive SGs after nerve crush coincided with those reactive after 5 min of pilo-
J.J. Vilches et al.r Journal of the Autonomic NerÕous System 74 (1998) 134–142
carpine injection in preoperative control molds. This fact suggests that these glands are hypersensitive to cholinergic stimulation. The ordered pharmacological activation of the number of SGs in a dose-dependent manner is known as recruitment ŽCollins et al., 1959; Kennedy et al., 1987., a phenomenon that can be attributed to the individual variations in sensitivity of SGs reported by Sato and Sato ŽSato and Sato, 1983.. In agreement with this pattern of response, the first secreting SGs are the most sensitive, while other SGs are progressively recruited as the periglandular concentration of neurotransmitter increases. Moreover, the first reactive SGs also maintain secretion over longer times, being still secreting 1 h after cholinergic stimulation ŽDole and Thaysen, 1953.. All these findings may explain the sustained activity over time shown by the reactive SGs after nerve crush and DDP intoxication, if the least sensitive glands are preferentially denervated. Although changes in SG responses to cholinergic agonists in nerve lesions and peripheral neuropathies have been widely reported previously, little was known about the sudomotor response to cholinergic antagonists in processes that cause postganglionic denervation. Our results prove that SGs develop atropine resistance that progress in parallel with the reduction to pilocarpine responsiveness. This fact might suggest that atropine resistance of SGs is directly related to the degree of denervation. Atropine is a competitive antagonist that produces its effects by occupying the muscarinic binding sites. Since the affinity of muscarinic binding sites is unaltered after denervation ŽGrant et al., 1991., the resistance to atropine might be a consequence of disregulation of SG responsiveness to cholinergic agonists produced by denervation. Changes in the cholinergic responses of partially denervated SGs such as resistance to atropine and oversustained activity, could be ascribed to a compensatory effect in order to counteract the sweating deficiency due to the existence of a number of unresponsive denervated SGs. It may be concluded that processes that cause postganglionic denervation of SGs modify the cholinergic response pattern of denervated SGs, and that these changes are proportional to the degree of denervation. Although completely denervated SGs become unresponsive to cholinergic stimulation, partially denervated SGs remain sensitive and have abnormal responses such as sustained secretion, decreased sweat output and partial resistance to atropine. The atropine resistance of SGs was the first sign of sudomotor impairment in DDP-induced neuropathy, followed by a decrease in the sweat output per gland and finally a decline in the number of reactive SGs. If further studies prove that DDP causes involvement of sudomotor axons, then, the sequence of abnormal sudomotor responses described above could be attributed to a process of denervation. Thus, detailed analyses of sudomotor responses to cholinergic agonists and antagonists allow to detect intermediate states of innervation with noninvasive methods and may be useful for the early detection of
141
denervation in peripheral neuropathies and nerve lesions, both in clinical and experimental research. Acknowledgements The authors greatly appreciate Monica Espejo and Jordi ´ Bruna for technical help, Kerr for providing Elasticon material, and Bristol-Myers Squibb for providing Neoplatin. This research was supported by grants from the FISSS Ž95-0033-02. and the CICYT ŽSAF97-0147., Spain. References Apfel, S.C., Arezzo, J.C., Lipson, L., Kessler, J.A., 1992. Nerve growth factor prevents experimental cisplatin neuropathy. Ann. Neurol. 31, 76–80. Barajon, I., Bersani, M., Quartu, M., Delfiacco, M., Cavaletti, G., Holst, J.J., Tredici, G., 1996. Neuropeptides and morphological changes in cisplatin-induced dorsal root ganglion neuronopathy. Exp. Neurol. 138, 93–104. Bharali, L.A.M., Burgess, S.A., Lisney, S.J.W., Pearson, D., 1988. Reinnervation of sweat glands in the rat hind paw following peripheral nerve injury. J. Auton. Nerv. Syst. 23, 125–129. Boogerd, W., Bokkel Huinink, W.W., Dalesio, O., Hoppenbrouwers, W.J.J.F., Jacob van der Sande, J., 1990. Cisplatin induced neuropathy: central, peripheral and autonomic nerve involvement. J. Neurooncol. 9, 255–263. Bridge, P.M., Ball, D.J., Mackinnon, S.E., Nakao, Y., Brandt, K., Hunter, D.A., Hertl, C., 1994. Nerve crush injuries—a model for axonotmesis. Exp. Neurol. 127, 284–290. Cannon, W.B., 1939. A law of denervation. Am. J. Med. Sci. 198, 739–750. Cardone, C., Dyck, P.J., 1990. A neuropathic deficit, decreased sweating, is prevented and ameliorated by euglycemia in streptozocin diabetes in rats. J. Clin. Invest. 86, 248–253. Cavaletti, G., Tredici, G., Marmiroli, P., Petruccioli, M.G., Barajon, I., Fabbrica, D., 1992. Morphometric study of the sensory neuron and peripheral nerve changes induced by chronic cisplatin ŽDDP. administration in rats. Acta Neuropathol. 84, 364–371. Collins, K.J., Sargent, F., Weiner, J.S., 1959. Excitation and depression of eccrine sweat glands by acetylcholine, acetyl-b-methylcholine and adrenaline. J. Physiol. 148, 592–614. Dale, H.H., Feldberg, W., 1934. The chemical transmission of secretory impulses to the sweat glands of the cat. J. Physiol. 82, 121–127. Dobson, R.L., Sato, K., 1972. The stimulation of eccrine sweating by pharmacologic agents. In: Montagna, W., Stoughton, R.B., Van Scott, E.J. ŽEds.., Advances in Biology of Skin, Appleton-Century-Crafts, New York, 1972, pp. 447–475. Dole, V.P., Thaysen, J.H., 1953. Variation in the functional power of human sweat glands. J. Exp. Med. 98, 129–143. Gao, W.-Q., Dybdal, N., Shinsky, N., Murnane, A., Schmelzer, C., Siegel, M., Keller, F., Phillips, H.S., Winslow, J.W., 1995. Neurotrophin-3 reverses experimental cisplatin-induced peripheral sensory neuropathy. Ann. Neurol. 38, 30–37. Grant, M.P., Landis, S.C., 1991. Developmental expression of muscarinic cholinergic receptors and coupling to phospholipase C in rat sweat glands are independent of innervation. J. Neurosci. 11, 3772–3782. Grant, M.P., Landis, S.C., Siegel, R.E., 1991. The molecular and pharmacological properties of muscarinic cholinergic receptors expressed by rat sweat glands are unaltered by denervation. J. Neurosci. 11, 3763–3771. Haftek, J., Thomas, P.K., 1968. Electron-microscope observations of the effects of localized crush injuries on the connective tissues of peripheral nerve. J. Anat. 103, 233–243.
142
J.J. Vilches et al.r Journal of the Autonomic NerÕous System 74 (1998) 134–142
Hayakawa, K., Sobue, G., Itoh, T., Mitsuma, T., 1994. Nerve growth factor prevents neurotoxic effects of cisplatin, vincristine and taxol, on adult rat sympathetic ganglion explants in vitro. Life Sci. 55, 519–525. Kennedy, W.R., Sakuta, M., 1984. Collateral reinnervation of sweat glands. Ann. Neurol. 15, 73–78. Kennedy, W.R., Navarro, X., 1989. Sympathetic sudomotor function in diabetic neuropathy. Arch. Neurol. 46, 1182–1186. Kennedy, W.R., Navarro, X., 1993. Evaluation of sudomotor function by sweat imprint methods. In: Low, P.A. ŽEd.., Evaluation and Management of Clinical Autonomic Disorders, Little and Brown, Boston, MA, 1993, pp. 253–261. Kennedy, W.R., Sakuta, M., Quick, D.C., 1984. Rodent eccrine sweat glands: a case of multiple efferent innervation. Neuroscience 11, 741–749. Kennedy, W.R., Sakuta, M., Sutherland, D., Goetz, F.C., 1984a. Quantitation of sweating deficiency in diabetes mellitus. Ann. Neurol. 15, 482–488. Kennedy, W.R., Sakuta, M., Sutherland, D., Goetz, F.C., 1984b. The sweating deficiency in diabetes mellitus: methods of quantitation and clinical correlation. Neurology 34, 758–763. Kennedy, W.R., Navarro, X., Gramprie, J., Quick, D.C., Sakuta, M., 1987. Orderly recruitment of sweat gland secretion. Ann. Neurol. 22, 150. Kihara, M., Opfer-Gehrking, T.L., Low, P.A., 1993. Comparison of directly stimulated with axon-reflex-mediated sudomotor responses in human subjects and in patients with diabetes. Muscle Nerve 16, 655–660. Levy, D.M., Reid, G., Abraham, R.R., Rowley, D.A., 1991. Assessment of basal and stimulated sweating in diabetes using a direct-reading computerized sudorometer. Diabetes Med. 8, S78–S81. List, C.F., Peet, M.M., 1938. Sweat secretion in man: III. Clinical observations on sweating produced by pilocarpine and mecholyl. Arch. Neurol. Psychiatr. 40, 268–290. Low, P.A., Caskey, P.E., Tuck, R.R., Fealey, R.D., Dyck, P.J., 1983. Quantitative sudomotor axon reflex test in normal and neuropathic subjects. Ann. Neurol. 14, 573–580. MacMillan, A.L., Spalding, J.M.K., 1969. Human sweating response to electrophoresed acetylcholine: a test of postganglionic sympathetic function. J. Neurol. Neurosurg. Psychiatr. 32, 155–160. Minor, V., 1927. Ein neues verfahren zu der klinischen untersuchung der schweißadsonderung. Zentralbl. Ges. Psysch. 47, 800–803. Mira, J.C., Fardeau, M., 1978. Nerve and muscle changes induced by repeated localized freezings of the sciatic nerve in the rat. In: Canal, N., Pozza, G. ŽEds.., Peripheral Neuropathies, Elsevier, Amsterdam, pp. 83–90. Navarro, X., Kennedy, W.R., 1989. Sweat gland reinnervation by sudomotor regeneration after different types of lesions and graft repairs. Exp. Neurol. 104, 229–234. Navarro, X., Kamei, H., Kennedy, W.R., 1988. Effect of age and maturation on sudomotor nerve regeneration in mice. Brain Res. 447, 133–140. Navarro, X., Miralles, R., Espadaler, J.M., Rubies-Prat, J., 1993a. Com´ parison of sympathetic sudomotor and skin responses in alcoholic neuropathy. Muscle Nerve 16, 404–407. Navarro, X., Verdu, E., 1993b. ´ E., Guerrero, J., Butı, ´ M., Gonalons, ˜ Abnormalities of sympathetic sudomotor function in experimental acrylamide neuropathy. J. Neurol. Sci. 114, 56–61. Navarro, X., Verdu, ´ E., Butı, ´ M., 1994. Comparison of regenerative and
reinnervating capabilities of different types of nerve fibers. Exp. Neurol. 129, 217–224. Nilvebrant, L., Ekstrom, J., Malmberg, L., 1986. Muscarinic receptor density in the urinary bladder after denervation, hypertrophy and urinary diversion. Acta Pharmacol. Toxicol. ŽCopenhagen. 59, 306– 314. Roelofs, R.I., Hrushesky, W., Rogin, J., Rosenberg, L., 1984. Peripheral sensory neuropathy and cisplatin chemotherapy. Neurology 34, 934– 938. Rosenfeld, C.S., Broder, L.E., 1984. Cisplatin-induced autonomic neuropathy. Cancer Treat. Rep. 68, 659–660. Russell, J.W., Windebank, A.J., McNiven, M.A., Brat, D.J., Brimijoin, W.S., 1995. Effect of cisplatin and ACTH4-9 on neural transport in cisplatin induced neurotoxicity. Brain Res. 676, 258–267. Sachs, D.J., Kloog, Y., Korczyn, A.D., Heron, D.S., Sokolovsky, M., 1979. Denervation, supersensitivity and muscarinic receptors in the cat iris. Biochem. Pharmacol. 28, 1513–1518. Sato, K., Sato, F., 1983. Individual variations in structure and function of human eccrine sweat gland. Am. J. Physiol. 245, R203–R208. Schmidt, Y., Unger, J.W., Bartke, I., Reiter, R., 1995. Effect of nerve growth factor on peptide neurons in dorsal root ganglia after taxol or cisplatin treatment and in diabetic Ždbrdb. mice. Exp. Neurol. 132, 16–23. Stevens, L.M., Landis, S.C., 1987. Development and properties of the secretory response in rat sweat glands: relationship to the induction of cholinergic function in sweat gland innervation. Dev. Biol. 123, 179–190. Stewart, J.D., Nguyen, D.M., Abrahamowicz, M., 1994. Quantitative sweat testing using acetylcholine for direct and axon reflex mediated stimulation with silicone mold recording; controls versus neuropathic diabetics. Muscle Nerve 17, 1370–1377. Talamo, B.R., Adler, S.C., Burt, D.R., 1979. Parasympathetic denervation decreases muscarinic receptor binding in rat parotid. Life Sci. 24, 1573–1581. Thompson, S.W., Davis, L.A.E., Kornfeld, M., Hilgers, R.D., Standefer, J.C., 1984. Cisplatin neuropathy: clinical, electrophysiologic, morphologic and toxicologic studies. Cancer 54, 1269–1275. Tomiwa, K., Nolan, C., Cavanagh, J.B., 1986. The effects of cisplatin on rat spinal ganglia: a study by light and electron microscopy and by morphometry. Acta Neuropathol. ŽBerlin. 69, 295–308. Tredici, G., Cavaletti, G., Petruccioli, M.G., Fabbrica, D., Pizzini, G., 1993. Degenerative processes in ganglionic neurons of the superior cervical ganglion in cisplatin-treated rats. J. Auton. Nerv. Syst. 43, 111. Verdu, ´ E., Navarro, X., 1997. Comparison of immunohistochemical and functional reinnervation of skin and muscle after peripheral nerve injury. Exp. Neurol. 146, 187–196. Verdu, ´ E., Butı, ´ M., Navarro, X., 1995. The effect of aging on efferent nerve fibers regeneration in mice. Brain Res. 696, 76–82. Verdu, ´ E., Butı, ´ M., Navarro, X., 1996. Functional changes of the peripheral nervous system with aging in the mouse. Neurobiol. Aging 17, 73–77. Verdu, F.J., Ceballos, D., Valero, A., ´ E., Vilches, J.J., Rodrıguez, ´ Navarro, X., 1998. Physiological and immunohistochemical characterization of cisplatin-induced neuropathy in mice, Muscle Nerve Žin press.. Vilches, J.J., Navarro, X., Verdu, ´ E., 1995. Functional sudomotor responses to cholinergic agonists and antagonists in the mouse. J. Auton. Nerv. Syst. 55, 105–111.
Changes in cholinergic responses of sweat glands during denervation and reinnervation Jorge J. Vilches, Francisco J. Rodrıguez, Enrique Verdu, ´ ´ Antoni Valero, Xavier Navarro
)
Department of Cell Biology and Physiology, Unitat de Fisiologia, Faculty of Medicine, UniÕersitat Autonoma de Barcelona, E-08193 Bellaterra, ` Barcelona, Spain Received 4 May 1998; revised 3 August 1998; accepted 9 September 1998
Abstract Functional sudomotor responses have been studied in sweat glands reinnervated after sciatic nerve crush and partially denervated by cisplatin intoxication in the mouse. The sudomotor function mediated by the sciatic nerve was evaluated by silicone imprints on the plantar surface of the hindpaws. Five days after nerve crush, completely denervated sweat glands became unresponsive to cholinergic stimulation with pilocarpine. During the following weeks, the number of reinnervated, reactive sweat glands increased progressively to reach a maximum of 89% of preoperative control counts by 40 days after nerve crush. At this time, the mean volume of sweat secreted per gland was normal, but reinnervated glands showed a secretory activity abnormally sustained over time after pilocarpine stimulation and, on the other hand, had an increased resistance to the inhibition of secretion induced by atropine. The effects of cisplatin administration on sudomotor function were investigated in two groups of mice, one treated with high doses of cisplatin Ž10 mgrkgrweek for 4 weeks. and another treated with low doses of cisplatin Ž5 mgrkgrweek for 8 weeks.. Cisplatin intoxication produced abnormal sudomotor responses indicative of denervation from cumulative doses of 10 mgrkg. The first abnormality found was a partial resistance of sweat glands to atropine, followed by a decrease in the sweat output per gland and finally a decline in the number of sweat glands activated by pilocarpine. These abnormalities in the sudomotor responses were more pronounced in mice treated with a high dose than in those with a lower dose regime. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Sweat glands; Cholinergic stimulation; Autonomic neuropathy; Denervation; Nerve lesion; Cisplatin
1. Introduction Sweat glands ŽSGs. are innervated by sympathetic postganglionic axons, that in contrast to the ordinary sympathetic innervation, use acetylcholine as the principal neurotransmitter ŽDale and Feldberg, 1934; Dobson and Sato, 1972; Stevens and Landis, 1987.. The cutaneous location of the SGs makes it possible to evaluate the functional activity of the sympathetic division of the autonomic nervous system with noninvasive methods such as colorimetric, evaporimetric and imprint techniques ŽMinor, 1927; Low et al., 1983; Kennedy et al., 1984a,b; Kennedy and Navarro, 1993.. Experimental evaluation of sudomotor function in intact animals has become possible by silastic imprints ŽKennedy and Sakuta, 1984; Kennedy and Navarro, 1993.. This method allows an accurate quantita) Corresponding author. Tel.: q34-9358-11966; fax: q34-9358-12986; e-mail: [email protected]
tion of the SG secretion, repeatedly over time, in order to follow changes of sudomotor function that occur either during natural processes such as maturation and aging ŽNavarro et al., 1988; Verdu´ et al., 1996., or after different types of nerve lesions ŽKennedy and Sakuta, 1984; Bharali et al., 1988; Navarro and Kennedy, 1989; Cardone and Dyck, 1990; Verdu´ et al., 1995.. SGs are apparent exceptions to the law of denervation hypersensitivity proposed by Cannon Ž1939.. Following an acute nerve lesion, denervated SGs become unresponsive to cholinergic agonists ŽList and Peet, 1938; MacMillan and Spalding, 1969; Kennedy and Sakuta, 1984; Navarro et al., 1988.. In previous reports we showed that activation of secretion of denervated SGs by pilocarpine was completely absent within 1 week after crushing or freezing the sciatic nerve of rodents. Reactive SGs first reappeared by 18–23 days after such nerve injuries and increased progressively in number to reach a maximum of approximately 90% of the pre-lesion baseline values at day 40,
0165-1838r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 1 8 3 8 Ž 9 8 . 0 0 1 5 2 - 0
J.J. Vilches et al.r Journal of the Autonomic NerÕous System 74 (1998) 134–142
135
Fig. 1. Silicone molds of the plantar surface of a mouse showing the sweat impressions obtained Ža. in a baseline preoperative test and Žb. 7 days after sciatic nerve crush and saphenectomy.
then remained about the same 2 months later ŽNavarro et al., 1988; Navarro et al., 1994; Verdu´ et al., 1995; Verdu´ and Navarro, 1997.. The progress of sudomotor nerve regeneration was judged by the reappearance of secreting SGs over time, thus, concluding that functional reinnervation was completed in the mouse by 40 days after nerve lesion. However, partially denervated SGs remain sensitive to pilocarpine ŽKennedy and Sakuta, 1984; Kennedy et al., 1984a,b.. This means that silastic imprints of SG secretion induced by cholinergic agents do not detect intermediate stages of sudomotor denervation. If more about the sudomotor responses to cholinergic agonists and antagonists was known, then partial innervation of SGs might become detectable using these techniques. Cisplatin Ž cis-dichlorodiammineplatinum II, DDP. is an antineoplastic drug widely used in the treatment of many common tumors including those affecting ovary, testes and
bladder. The compound has several toxic side-effects, but at present, neurotoxicity is considered to be the major dose-limiting effect. DDP causes a predominantly sensory peripheral neuropathy ŽRoelofs et al., 1984; Thompson et al., 1984; Boogerd et al., 1990. with occasional autonomic dysfunctions ŽRosenfeld and Broder, 1984; Boogerd et al., 1990.. Cisplatin administration to rats under different dose regimes induces early morphological changes in dorsal root ganglia neurons ŽTomiwa et al., 1986; Cavaletti et al., 1992., supporting the hypothesis that DDP induces a primary neuronopathy of dorsal root ganglia neurons followed by a mild axonopathy mainly involving large myelinated fibers ŽCavaletti et al., 1992; Gao et al., 1995; Barajon et al., 1996.. Similar pathological changes have been described in ganglionic sympathetic neurons ŽTredici et al., 1993., that may underlie the autonomic disfunctions reported following DDP therapy. The aim of the present study was to evaluate the functional changes of cholinergic sudomotor responses in Table 1 Comparison of the total number of reactive sweat glands ŽNo SGs. and the sweat output per gland ŽSOrGS. 10 min after pilocarpine injection, and percentage of SGs reactive to pilocarpine 20 min after atropine administration Ž% SGs. in the operated hindpaw 40 days after a sciatic nerve crush ŽCX. and in the contralateral intact hindpaw ŽCTL. from the same mice Paw
Parameter No SGs
CX CTL Fig. 2. Total number of reactive sweat glands ŽNo SGs. in the hindpaw 10 min after pilocarpine injection during the reinnervation process after sciatic nerve crush. Values are expressed as means and S.E.M.
296.6"12.5 365.5"18.5
a
SOrSG Žnl.
% SGs
0.22"0.02 0.21"0.01
91.6"3.3 a 49.4"9.7
Values are expressed as means"S.E.M. a p- 0.05 vs. CTL.
136
J.J. Vilches et al.r Journal of the Autonomic NerÕous System 74 (1998) 134–142
denervation and reinnervation processes. In order to assess the time course and severity of functional abnormalities, we made serial testing after either an acute nerve lesion by crushing the sciatic nerve or different cumulative doses of DDP. 2. Materials and methods 2.1. Experimental design Four groups of Swiss female mice, aged 2.5 months at the beginning of the experiments, were used. Animals of
Fig. 4. Total number of reactive sweat glands ŽNo SGs. after pilocarpine injection in groups HD Žtop., LD Žmiddle. and CTL Žbottom.. Cumulative doses of DDP in the legend are expressed as mgrkg. Values are shown as means and S.E.M.
Fig. 3. ŽA. Total number of reactive sweat glands ŽNo SGs. in the hindpaw and ŽB. sweat output per gland ŽSOrSG. after pilocarpine injection 40 days after sciatic nerve crush, in the operated hindpaw ŽCX. and in the contralateral intact hindpaw ŽCTL. from the same mice. Values are shown as means and S.E.M.; time is in minutes after pilocarpine injection. ŽC. Effects of atropine on the glandular stimulation induced by pilocarpine 43 days after sciatic nerve crush with doses of atropine 0.05 mgrkg ŽCX 0.05. or 0.1 mgrkg ŽCX 0.1. and 0.05 mgrkg in the intact hindpaw ŽCTL 0.05. from the same mice. Values are expressed as means and S.E.M. of the percentage of reactive sweat glands Ž% SGs. with respect to the total number responsive to pilocarpine. Time is in minutes after atropine administration.
one group Ž n s 10. were subjected to a sciatic nerve crush. Under pentobarbital anesthesia Ž50 mgrkg, i.p.., the saphenous nerve was cut in the femoral space and a long segment of the distal stump removed to prevent regeneration. The sciatic nerve was then exposed and crushed three times in succession with a fine forceps ŽDumont no. 5. at 45 mm from the tip of the third digit. Finally, the skin was closed with 4-0 sutures and disinfected with povidone iodine solution. The number of reactive SGs 10 min after pilocarpine injection Ž5 mgrkg, s.c.. was evaluated before operation and at several intervals up to 90 days post-operation. Since by 40 days after sciatic crush the number of SGs reactive to pilocarpine achieves maximal values, similar to pre-operative controls ŽNavarro and Kennedy, 1989; Verdu´ et al., 1995., we made specific tests in order to detect changes in the cholinergic responses by 40 and 60 days. As the number of SGs on right and left paws are essentially equal ŽNavarro et al., 1988., these tests were also done in the contralateral paw as control.
J.J. Vilches et al.r Journal of the Autonomic NerÕous System 74 (1998) 134–142
In the second experimental paradigm three groups of mice were used. One group ŽHD, n s 10. was injected with DDP ŽNeoplatin w , Bristol-Myers Squibb. dissolved in sterile saline solution, 10 mgrkg i.p. once a week for 4 weeks. This group was evaluated before the onset of treatment and biweekly at cumulative doses of 20 and 40 mgrkg. A second group ŽLD, n s 9. was given DDP at a lower dose, 5 mgrkg i.p. once a week for 8 weeks. This group was evaluated before the onset of DDP treatment and biweekly at cumulative doses of 10, 20, 30 and 40 mgrkg. To prevent DDP-induced renal damage, 1 ml of saline solution was injected subcutaneously immediately after DDP administration. In a control group ŽCTL, n s 8. animals were treated with saline intraperitoneally once a week for 8 weeks. 2.2. Functional tests The number of secreting SGs and the sweat output per gland was determined by making an impression mold of the plantar surface of the hindpaw using a silicone material ŽElasticon, Kerr, Romulus, MI.. A mixture of 0.2 ml base material plus four drops of hardener from a 23-gauge needle was gently spread with a spatula over the sole. As the silicone hardened Žduring approximately 3 min., it retained the impressions of the sweat droplets as they emerged from the sweat ducts and pushed up into the mold. Each indentation has been shown by histological methods to mark the location of one sweat duct ŽKennedy et al., 1984a,b.. Droplet impressions were counted under a dissecting microscope Ž=20. using transmitted light. Total counts of SGs were made for each paw and for defined subdivisions of the paw ŽKennedy and Sakuta, 1984; Kennedy et al., 1984a,b.. The diameter of each sweat impression in the third toe flat area was measured using a micrometer grid, and used to calculate the volume of sweat
137
droplets considered to approximate a sphere Žvolumes 4r3 p r 3 . ŽKennedy et al., 1984a,.. For the assessment of sudomotor responses to cholinergic stimulation, pilocarpine nitrate Ž5 mgrkg. was injected subcutaneously into the back of the mice, as this dose of pilocarpine produces a maximal and stable sudomotor response in the mouse ŽVilches et al., 1995.. Silicone molds were made 5, 10, 15, 20, 30 and 45 min after injection. For evaluating the antagonistic inhibition of sweating, a dose of 0.05 mgrkg of atropine was injected subcutaneously 15 min after pilocarpine injection. Silicone molds were made 10 min after pilocarpine injection Žmaximal number of reactive SGs. and 5, 10, 15 and 20 min after atropine administration. In order to abolish emotional or basal sweating, the distal innervation of the paw was blocked by injecting 0.05 ml of bupivacaine Ž0.75%. at the ankle before the pilocarpine injection ŽVilches et al., 1995.. Values of sudomotor responses to pilocarpine stimulation are shown as the absolute number of sweat impressions in the mold and the estimated sweat output per SG, whereas results obtained after atropine administration are expressed as the percentage of the number of impressions found 10 min after pilocarpine injection. All results are shown as means and S.E.M. Statistical comparisons between values obtained from different groups of mice were made by ANOVA with the Scheffe´ test for multiple comparisons, and comparisons between values obtained from the same mice by the paired t-test.
3. Results 3.1. Sudomotor responses after nerÕe crush After sciatic nerve crush, the number of plantar SGs activated by pilocarpine declined daily and reached zero
Table 2 Results of the total number of reactive SGs ŽNo SGs. and the sweat output per gland ŽSOrSG. 10 min after pilocarpine injection, and percentage of SGs reactive to pilocarpine Ž% SGs. 20 min after atropine administration in control mice ŽCTL. and mice treated with low ŽLD. and high ŽHD. doses of DDP Parameter
No SGs
SOrSG Žnl.
% SGs
Group
CTL LD HD CTL LD HD CTL LD HD
Cumulative dose Žmgrkg. Baseline
10
20
30
40
400.5 " 11.0 404.8 " 10.5 420.3 " 10.5 0.15 " 0.02 0.16 " 0.02 0.14 " 0.02 23.8 " 11.2 7.0 " 1.9 25.8 " 14.2
413.0 " 12.6 415.3 " 14.3
415.6 " 10.7 405.3 " 14.8 358.7 " 25.2 0.17 " 0.02 0.16 " 0.02 0.13 " 0.02 37.2 " 11.7 46.3 " 9.8 c 86.9 " 6.0 a,b,c
411.9 " 8.4 389.7 " 15.8
406.3 " 9.0 306.4 " 37.0 a,c 148.1 " 44.8 a,b,c 0.17 " 0.03 0.08 " 0.01a,c 0.03 " 0.01a,b,c 20.9 " 8.0 69.8 " 10.6 a,c 91.3 " 5.1 a
0.17 " 0.02 0.19 " 0.01 39.6 " 7.7 53.3 " 10.8 c
0.16 " 0.02 0.11 " 0.01a,c 41.7 " 9.6 77.7 " 4.9 a,c
Values are expressed as means" S.E.M. n s 8, 9 and 10 for groups CTL, LD and HD respectively, except for the test of atropine inhibition at a cumulative dose of 40 mgrkg, at which the number of mice was 8, 6 and 4 for groups CTL, LD and HD, respectively. a p - 0.05 vs. CTL. b p - 0.05 vs. LD. c p - 0.05 vs. baseline values obtained before the onset of treatment.
138
J.J. Vilches et al.r Journal of the Autonomic NerÕous System 74 (1998) 134–142
response in reinnervated SGs ŽTable 1.. However, a higher dose of 0.1 mgrkg atropine produced a significant reduction of the percentage of secreting SGs in the operated paw ŽFig. 3C.. Similar results were obtained at postoperative days 60 and 63, in the SG responses to pilocarpine as well as in the inhibition induced by atropine. 3.2. Sudomotor responses during DDP intoxication Fig. 4 shows the number of SGs responsive to pilocarpine in HD, LD and CTL groups. The highest number of reactive SGs was observed 10 min after pilocarpine administration in the three groups of mice. The number of reactive SGs decreased progressively with increasing cumulative doses of DDP in treated groups, more markedly in group HD than in group LD, although the maximal number of reactive SGs was significantly lower with re-
Fig. 5. Sweating response in two footpads of a mouse from the LD group Ža. before the onset of DDP treatment and Žb. at a 40 mgrkg cumulative dose.
by day 5 ŽFig. 1.. The first reactive SGs appeared two weeks after lesion and their number increased rapidly to reach a maximum of 89% of the preoperative values at 40 days postoperation. This number did not increase at 60 and 90 days tests ŽFig. 2.. Despite the fact that 40 days after nerve lesion the maximum number of reactive SGs in the denervated paw was lower than in the intact contralateral paw of the same mice, this difference was only observed 10 min after pilocarpine injection ŽTable 1.. In the following molds the number of reactive SGs in the reinnervated paw did not differ significantly from those in the contralateral paw, due to the slight decline in the number of secreting SGs in the control paw in contrast to the stable response of the operated paw ŽFig. 3A.. On the contrary, 40 days after nerve crush the sweat output per gland was similar in both paws; furthermore, 45 min after pilocarpine injection the volume secreted by reinnervated SGs was slightly larger than that produced by control SGs ŽFig. 3B.. The reinnervated SGs showed a partial resistance to atropine 43 days after nerve crush. Whereas in the control paw a dose of 0.05 mgrkg of atropine produced a marked decline in the percentage of SGs reactive to pilocarpine, the same dose did not blocked the pilocarpine-induced
Fig. 6. Sweat output per gland ŽSOrSG. after pilocarpine injection in groups HD Žtop., LD Žmiddle. and CTL Žbottom.. Cumulative doses of DDP in the legend are expressed in mgrkg. Values are shown as means and S.E.M.
J.J. Vilches et al.r Journal of the Autonomic NerÕous System 74 (1998) 134–142
spect to controls only at a 40 mgrkg cumulative dose ŽTable 2, Fig. 5.. The number of reactive SGs to pilocarpine tended to be abnormally sustained from 10 to 45 min after injection in DDP-treated mice, while in control mice there was a decline most evident between 10 and 20 min molds. Similar findings were observed for the sweat output per gland ŽFig. 6.. The maximal volume secreted per reactive SGs was achieved 10 min after pilocarpine administration, both in control and DDP-treated mice. The sweat output per gland also decreased progressively with increasing cumulative doses, more in group HD than in group LD. The decrease in the sweat volume was more pronounced than the decline in number of secreting SGs, reaching a significant difference with respect to controls by cumulative doses of 30 mgrkg ŽTable 2..
Fig. 7. Effects of atropine on the SG stimulation induced by pilocarpine in groups HD Žtop., LD Žmiddle. and CTL Žbottom.. Cumulative doses of DDP in the legend are expressed in mgrkg. Values are shown as means and S.E.M. of the percentage of reactive sweat glands Ž% SGs. with respect to the number responsive to pilocarpine. Time is in minutes after atropine administration.
139
Atropine produced a strong reduction in the percentage of SGs reactive to pilocarpine in group CTL as well as in the baseline results of groups HD and LD mice. However, DDP intoxication caused an increased resistance of reactive SGs to atropine, that was more evident in group HD than in group LD ŽFig. 7.. The resistance to atropine was statistically significant from cumulative doses of 10 mgrkg ŽTable 2. and tended to increase at higher doses, while the results of the control group did not change notably throughout the follow-up, neither for pilocarpine-induced responses nor for atropine inhibition.
4. Discussion The insensitivity of denervated SGs to cholinergic agonists provides a convenient system to evaluate the degree of functional innervation of SGs by sudomotor sympathetic nerves in situations that may cause denervation and reinnervation processes ŽKennedy and Navarro, 1993.. By further investigating the sudomotor responses to cholinergic agonists and antagonists, we have found that SGs which are presumably only partially innervated, as a consequence of reinnervation after nerve injury, show abnormal responses, such as a more sustained secretion over time after stimulation and a partial resistance to the inhibitory effect of atropine. Denervation of SGs as a consequence of traumatic peripheral nerve lesions lead to their lack of reactivity to cholinergic stimuli. After complete axotomy by sectioning, crushing or freezing the mouse sciatic nerve the number of secreting SGs decreases exponentially to zero during the first week ŽKennedy and Sakuta, 1984; Navarro et al., 1988; Navarro and Kennedy, 1989., as also confirmed in the present study. Sudomotor regeneration, judged by the reappearance of pilocarpine-reactive SGs, was progressive and reached the maximum by 40 days after nerve crush, in agreement with the results of previous reports ŽNavarro and Kennedy, 1989; Navarro et al., 1994; Verdu´ et al., 1995; Verdu´ and Navarro, 1997.. Focal lesions produced by freezing or crushing peripheral nerves cause complete axotomy ŽBridge et al., 1994., but do not disrupt the basal lamina surrounding each nerve fiber or the epineural sheaths ŽHaftek and Thomas, 1968; Mira and Fardeau, 1978., thus allowing an efficient regeneration and an appropriate reinnervation of target organs. The number of reinnervated reactive SGs after a crush injury reaches close to normal values. Nevertheless, in our study newly reinnervated SGs showed peculiar abnormal cholinergic responses. A sustained secretory activity was observed in reinnervated SGs, manifested by the stability in the number of reactive SGs and in the sweat output per gland over time after pilocarpine administration. This evidence is compatible with the long duration of SG reactivity reported by Kennedy and Sakuta ŽKennedy and Sakuta, 1984. a few hours after sciatic nerve section in the mouse,
140
J.J. Vilches et al.r Journal of the Autonomic NerÕous System 74 (1998) 134–142
as well as with the overrepresentation of large diameter droplets reported in diabetic patients with mild neuropathy ŽKihara et al., 1993.. On the other hand, SGs of the lesioned paw were more resistant to the inhibition induced by atropine than SGs of the contralateral intact paw. Although a positive correlation between functional sudomotor recovery and the density of PGP 9.5-immunoreactive nerve fibers around the SGs has been found after sciatic nerve crush in the mouse, the density of sudomotor fibers even 100 days postoperation was about 50% lower than in unoperated control samples ŽVerdu´ and Navarro, 1997.. This finding supports the view that partially innervated SGs remain responsive to cholinergic stimulation. Thus, the abnormal cholinergic responses of reinnervated SGs showed in the present study may be a functional evidence of a deficit in SG innervation. The results of this study also show that chronic administration of DDP induces a decline in the number of reactive SGs and the sweat output per gland. Functional disturbances developed progressively with higher cumulative doses and were more pronounced in mice treated with a high dose than in those with a lower dose regime. These findings are compatible with the lack of responsiveness of denervated SGs in nerve lesions and in toxic or metabolic neuropathies. In diabetic subjects, several studies have shown a decrease in the number of reactive SGs, the sweat rate and the sweat output after cholinergic stimulation ŽLow et al., 1983; Kennedy et al., 1984a,b; Kennedy and Navarro, 1989; Stewart et al., 1994.. Similar results have been reported in alcoholic neuropathy ŽNavarro et al., 1993a,b. and in experimental acrylamide intoxication ŽNavarro et al., 1993a,b.. All these findings suggest that DDP intoxication affects sympathetic postganglionic function. Morphological studies of peripheral nerves of DDPtreated rodents showed degenerative signs and loss of myelinated nerve fibers to a mild degree, more evident in distal than proximal nerves, and little or no change in unmyelinated fibers ŽCavaletti et al., 1992; Gao et al., 1995.. However, the similar ultrastructural changes observed in dorsal root and sympathetic ganglia neurons ŽTomiwa et al., 1986; Cavaletti et al., 1992; Tredici et al., 1993; Barajon et al., 1996. support the hypotheses that these cells are the primary targets of DDP-induced neurotoxicity, due to the absence of an efficient blood–nerve barrier at these two sites. Moreover, DDP inhibits the neurite outgrowth in cultures of rat superior cervical ganglion ŽHayakawa et al., 1994.. It has been reported that DDP reduces the levels of secretory neuropeptides both in dorsal root ganglia ŽApfel et al., 1992; Schmidt et al., 1995. and in the distal nerve fibers innervating the skin and the SGs ŽVerdu´ et al., 1998.. The loss of neurotransmitter supply, due to an impairment of the axonal transport ŽRussell et al., 1995., might lead to an early decline in sudomotor function, followed by later structural denervation ŽVerdu´ et al., 1998.. Nevertheless, changes in sudomotor responses of DDP-treated mice might be also due to
a direct toxic effect of DDP on SGs. Thus, further studies are necessary to determine the toxicity of DDP on SGs and postganglionic sudomotor neurons. In our study, the decrease in the number of reactive SGs was significant when reaching a DDP cumulative dose of 40 mgrkg. SGs that still remained reactive to pilocarpine showed an abnormally maintained response, similar to that found in reinnervated SGs after nerve crush. An excessive, prolonged sweating response has also been described in some diabetic patients after cholinergic stimulation ŽLow et al., 1983; Levy et al., 1991.. The decrease in the sweat output per gland was more marked and appeared earlier than the decline in number of reactive SGs. The partially denervated SGs would produce smaller sweat droplets, before ceasing secretion when completely denervated. This evidence is in agreement with findings from a large series of diabetic patients, in whom about a 15% had a reduced mean volume of sweat secretion with a normal number of reactive SGs ŽKennedy and Navarro, 1989.. On the other hand, an increased resistance of SGs to atropine inhibition was observed already at cumulative doses of 10 mgrkg, indicating that it may be considered as the first sign of sudomotor impairment in DDP-treated mice. This sudomotor abnormality even precedes the appearance of sensory nerve conduction slowing ŽVerdu´ et al., 1998.. Therefore, these findings would recommend to perform a thorough evaluation of sudomotor activity in patients treated with DDP, in order to detect its neurotoxicity at early stages. Relatively little is known about the loss of responsiveness of denervated SGs. The insensitivity of denervated SGs could be due to a decline in number or function of SG muscarinic receptors. The effect of cholinergic denervation on muscarinic receptor expression is diverse in other peripheral targets. Denervation of cat iris produces no change in the concentration or affinity of muscarinic receptors ŽSachs et al., 1979.. In contrast, parasympathectomy of the rat urinary bladder provokes an increase ŽNilvebrant et al., 1986., whereas denervation of rat parotid gland causes a decrease in the density of muscarinic receptors ŽTalamo et al., 1979.. However, in all these targets denervation produces hypersensitivity to cholinergic agonists, suggesting that regulation of the functional responsiveness does not depend of the receptor expression. The case of denervated SGs supports this hypothesis, as the density, the level of expression and the distribution of SG muscarinic receptors do not change 7 days after transection of the rat sciatic nerve ŽGrant et al., 1991.. Furthermore, the molecular subtype of muscarinic receptor and its affinity were also unchanged after denervation. Another explanation of nonresponsiveness is that the functional receptor was present but uncoupled from the secretory pathway. Since G-protein coupling is unaffected by denervation ŽGrant and Landis, 1991., the most likely cause of SGs insensitivity would be a change in a step of the pathway distal to the G-protein. We noticed that the first responsive SGs after nerve crush coincided with those reactive after 5 min of pilo-
J.J. Vilches et al.r Journal of the Autonomic NerÕous System 74 (1998) 134–142
carpine injection in preoperative control molds. This fact suggests that these glands are hypersensitive to cholinergic stimulation. The ordered pharmacological activation of the number of SGs in a dose-dependent manner is known as recruitment ŽCollins et al., 1959; Kennedy et al., 1987., a phenomenon that can be attributed to the individual variations in sensitivity of SGs reported by Sato and Sato ŽSato and Sato, 1983.. In agreement with this pattern of response, the first secreting SGs are the most sensitive, while other SGs are progressively recruited as the periglandular concentration of neurotransmitter increases. Moreover, the first reactive SGs also maintain secretion over longer times, being still secreting 1 h after cholinergic stimulation ŽDole and Thaysen, 1953.. All these findings may explain the sustained activity over time shown by the reactive SGs after nerve crush and DDP intoxication, if the least sensitive glands are preferentially denervated. Although changes in SG responses to cholinergic agonists in nerve lesions and peripheral neuropathies have been widely reported previously, little was known about the sudomotor response to cholinergic antagonists in processes that cause postganglionic denervation. Our results prove that SGs develop atropine resistance that progress in parallel with the reduction to pilocarpine responsiveness. This fact might suggest that atropine resistance of SGs is directly related to the degree of denervation. Atropine is a competitive antagonist that produces its effects by occupying the muscarinic binding sites. Since the affinity of muscarinic binding sites is unaltered after denervation ŽGrant et al., 1991., the resistance to atropine might be a consequence of disregulation of SG responsiveness to cholinergic agonists produced by denervation. Changes in the cholinergic responses of partially denervated SGs such as resistance to atropine and oversustained activity, could be ascribed to a compensatory effect in order to counteract the sweating deficiency due to the existence of a number of unresponsive denervated SGs. It may be concluded that processes that cause postganglionic denervation of SGs modify the cholinergic response pattern of denervated SGs, and that these changes are proportional to the degree of denervation. Although completely denervated SGs become unresponsive to cholinergic stimulation, partially denervated SGs remain sensitive and have abnormal responses such as sustained secretion, decreased sweat output and partial resistance to atropine. The atropine resistance of SGs was the first sign of sudomotor impairment in DDP-induced neuropathy, followed by a decrease in the sweat output per gland and finally a decline in the number of reactive SGs. If further studies prove that DDP causes involvement of sudomotor axons, then, the sequence of abnormal sudomotor responses described above could be attributed to a process of denervation. Thus, detailed analyses of sudomotor responses to cholinergic agonists and antagonists allow to detect intermediate states of innervation with noninvasive methods and may be useful for the early detection of
141
denervation in peripheral neuropathies and nerve lesions, both in clinical and experimental research. Acknowledgements The authors greatly appreciate Monica Espejo and Jordi ´ Bruna for technical help, Kerr for providing Elasticon material, and Bristol-Myers Squibb for providing Neoplatin. This research was supported by grants from the FISSS Ž95-0033-02. and the CICYT ŽSAF97-0147., Spain. References Apfel, S.C., Arezzo, J.C., Lipson, L., Kessler, J.A., 1992. Nerve growth factor prevents experimental cisplatin neuropathy. Ann. Neurol. 31, 76–80. Barajon, I., Bersani, M., Quartu, M., Delfiacco, M., Cavaletti, G., Holst, J.J., Tredici, G., 1996. Neuropeptides and morphological changes in cisplatin-induced dorsal root ganglion neuronopathy. Exp. Neurol. 138, 93–104. Bharali, L.A.M., Burgess, S.A., Lisney, S.J.W., Pearson, D., 1988. Reinnervation of sweat glands in the rat hind paw following peripheral nerve injury. J. Auton. Nerv. Syst. 23, 125–129. Boogerd, W., Bokkel Huinink, W.W., Dalesio, O., Hoppenbrouwers, W.J.J.F., Jacob van der Sande, J., 1990. Cisplatin induced neuropathy: central, peripheral and autonomic nerve involvement. J. Neurooncol. 9, 255–263. Bridge, P.M., Ball, D.J., Mackinnon, S.E., Nakao, Y., Brandt, K., Hunter, D.A., Hertl, C., 1994. Nerve crush injuries—a model for axonotmesis. Exp. Neurol. 127, 284–290. Cannon, W.B., 1939. A law of denervation. Am. J. Med. Sci. 198, 739–750. Cardone, C., Dyck, P.J., 1990. A neuropathic deficit, decreased sweating, is prevented and ameliorated by euglycemia in streptozocin diabetes in rats. J. Clin. Invest. 86, 248–253. Cavaletti, G., Tredici, G., Marmiroli, P., Petruccioli, M.G., Barajon, I., Fabbrica, D., 1992. Morphometric study of the sensory neuron and peripheral nerve changes induced by chronic cisplatin ŽDDP. administration in rats. Acta Neuropathol. 84, 364–371. Collins, K.J., Sargent, F., Weiner, J.S., 1959. Excitation and depression of eccrine sweat glands by acetylcholine, acetyl-b-methylcholine and adrenaline. J. Physiol. 148, 592–614. Dale, H.H., Feldberg, W., 1934. The chemical transmission of secretory impulses to the sweat glands of the cat. J. Physiol. 82, 121–127. Dobson, R.L., Sato, K., 1972. The stimulation of eccrine sweating by pharmacologic agents. In: Montagna, W., Stoughton, R.B., Van Scott, E.J. ŽEds.., Advances in Biology of Skin, Appleton-Century-Crafts, New York, 1972, pp. 447–475. Dole, V.P., Thaysen, J.H., 1953. Variation in the functional power of human sweat glands. J. Exp. Med. 98, 129–143. Gao, W.-Q., Dybdal, N., Shinsky, N., Murnane, A., Schmelzer, C., Siegel, M., Keller, F., Phillips, H.S., Winslow, J.W., 1995. Neurotrophin-3 reverses experimental cisplatin-induced peripheral sensory neuropathy. Ann. Neurol. 38, 30–37. Grant, M.P., Landis, S.C., 1991. Developmental expression of muscarinic cholinergic receptors and coupling to phospholipase C in rat sweat glands are independent of innervation. J. Neurosci. 11, 3772–3782. Grant, M.P., Landis, S.C., Siegel, R.E., 1991. The molecular and pharmacological properties of muscarinic cholinergic receptors expressed by rat sweat glands are unaltered by denervation. J. Neurosci. 11, 3763–3771. Haftek, J., Thomas, P.K., 1968. Electron-microscope observations of the effects of localized crush injuries on the connective tissues of peripheral nerve. J. Anat. 103, 233–243.
142
J.J. Vilches et al.r Journal of the Autonomic NerÕous System 74 (1998) 134–142
Hayakawa, K., Sobue, G., Itoh, T., Mitsuma, T., 1994. Nerve growth factor prevents neurotoxic effects of cisplatin, vincristine and taxol, on adult rat sympathetic ganglion explants in vitro. Life Sci. 55, 519–525. Kennedy, W.R., Sakuta, M., 1984. Collateral reinnervation of sweat glands. Ann. Neurol. 15, 73–78. Kennedy, W.R., Navarro, X., 1989. Sympathetic sudomotor function in diabetic neuropathy. Arch. Neurol. 46, 1182–1186. Kennedy, W.R., Navarro, X., 1993. Evaluation of sudomotor function by sweat imprint methods. In: Low, P.A. ŽEd.., Evaluation and Management of Clinical Autonomic Disorders, Little and Brown, Boston, MA, 1993, pp. 253–261. Kennedy, W.R., Sakuta, M., Quick, D.C., 1984. Rodent eccrine sweat glands: a case of multiple efferent innervation. Neuroscience 11, 741–749. Kennedy, W.R., Sakuta, M., Sutherland, D., Goetz, F.C., 1984a. Quantitation of sweating deficiency in diabetes mellitus. Ann. Neurol. 15, 482–488. Kennedy, W.R., Sakuta, M., Sutherland, D., Goetz, F.C., 1984b. The sweating deficiency in diabetes mellitus: methods of quantitation and clinical correlation. Neurology 34, 758–763. Kennedy, W.R., Navarro, X., Gramprie, J., Quick, D.C., Sakuta, M., 1987. Orderly recruitment of sweat gland secretion. Ann. Neurol. 22, 150. Kihara, M., Opfer-Gehrking, T.L., Low, P.A., 1993. Comparison of directly stimulated with axon-reflex-mediated sudomotor responses in human subjects and in patients with diabetes. Muscle Nerve 16, 655–660. Levy, D.M., Reid, G., Abraham, R.R., Rowley, D.A., 1991. Assessment of basal and stimulated sweating in diabetes using a direct-reading computerized sudorometer. Diabetes Med. 8, S78–S81. List, C.F., Peet, M.M., 1938. Sweat secretion in man: III. Clinical observations on sweating produced by pilocarpine and mecholyl. Arch. Neurol. Psychiatr. 40, 268–290. Low, P.A., Caskey, P.E., Tuck, R.R., Fealey, R.D., Dyck, P.J., 1983. Quantitative sudomotor axon reflex test in normal and neuropathic subjects. Ann. Neurol. 14, 573–580. MacMillan, A.L., Spalding, J.M.K., 1969. Human sweating response to electrophoresed acetylcholine: a test of postganglionic sympathetic function. J. Neurol. Neurosurg. Psychiatr. 32, 155–160. Minor, V., 1927. Ein neues verfahren zu der klinischen untersuchung der schweißadsonderung. Zentralbl. Ges. Psysch. 47, 800–803. Mira, J.C., Fardeau, M., 1978. Nerve and muscle changes induced by repeated localized freezings of the sciatic nerve in the rat. In: Canal, N., Pozza, G. ŽEds.., Peripheral Neuropathies, Elsevier, Amsterdam, pp. 83–90. Navarro, X., Kennedy, W.R., 1989. Sweat gland reinnervation by sudomotor regeneration after different types of lesions and graft repairs. Exp. Neurol. 104, 229–234. Navarro, X., Kamei, H., Kennedy, W.R., 1988. Effect of age and maturation on sudomotor nerve regeneration in mice. Brain Res. 447, 133–140. Navarro, X., Miralles, R., Espadaler, J.M., Rubies-Prat, J., 1993a. Com´ parison of sympathetic sudomotor and skin responses in alcoholic neuropathy. Muscle Nerve 16, 404–407. Navarro, X., Verdu, E., 1993b. ´ E., Guerrero, J., Butı, ´ M., Gonalons, ˜ Abnormalities of sympathetic sudomotor function in experimental acrylamide neuropathy. J. Neurol. Sci. 114, 56–61. Navarro, X., Verdu, ´ E., Butı, ´ M., 1994. Comparison of regenerative and
reinnervating capabilities of different types of nerve fibers. Exp. Neurol. 129, 217–224. Nilvebrant, L., Ekstrom, J., Malmberg, L., 1986. Muscarinic receptor density in the urinary bladder after denervation, hypertrophy and urinary diversion. Acta Pharmacol. Toxicol. ŽCopenhagen. 59, 306– 314. Roelofs, R.I., Hrushesky, W., Rogin, J., Rosenberg, L., 1984. Peripheral sensory neuropathy and cisplatin chemotherapy. Neurology 34, 934– 938. Rosenfeld, C.S., Broder, L.E., 1984. Cisplatin-induced autonomic neuropathy. Cancer Treat. Rep. 68, 659–660. Russell, J.W., Windebank, A.J., McNiven, M.A., Brat, D.J., Brimijoin, W.S., 1995. Effect of cisplatin and ACTH4-9 on neural transport in cisplatin induced neurotoxicity. Brain Res. 676, 258–267. Sachs, D.J., Kloog, Y., Korczyn, A.D., Heron, D.S., Sokolovsky, M., 1979. Denervation, supersensitivity and muscarinic receptors in the cat iris. Biochem. Pharmacol. 28, 1513–1518. Sato, K., Sato, F., 1983. Individual variations in structure and function of human eccrine sweat gland. Am. J. Physiol. 245, R203–R208. Schmidt, Y., Unger, J.W., Bartke, I., Reiter, R., 1995. Effect of nerve growth factor on peptide neurons in dorsal root ganglia after taxol or cisplatin treatment and in diabetic Ždbrdb. mice. Exp. Neurol. 132, 16–23. Stevens, L.M., Landis, S.C., 1987. Development and properties of the secretory response in rat sweat glands: relationship to the induction of cholinergic function in sweat gland innervation. Dev. Biol. 123, 179–190. Stewart, J.D., Nguyen, D.M., Abrahamowicz, M., 1994. Quantitative sweat testing using acetylcholine for direct and axon reflex mediated stimulation with silicone mold recording; controls versus neuropathic diabetics. Muscle Nerve 17, 1370–1377. Talamo, B.R., Adler, S.C., Burt, D.R., 1979. Parasympathetic denervation decreases muscarinic receptor binding in rat parotid. Life Sci. 24, 1573–1581. Thompson, S.W., Davis, L.A.E., Kornfeld, M., Hilgers, R.D., Standefer, J.C., 1984. Cisplatin neuropathy: clinical, electrophysiologic, morphologic and toxicologic studies. Cancer 54, 1269–1275. Tomiwa, K., Nolan, C., Cavanagh, J.B., 1986. The effects of cisplatin on rat spinal ganglia: a study by light and electron microscopy and by morphometry. Acta Neuropathol. ŽBerlin. 69, 295–308. Tredici, G., Cavaletti, G., Petruccioli, M.G., Fabbrica, D., Pizzini, G., 1993. Degenerative processes in ganglionic neurons of the superior cervical ganglion in cisplatin-treated rats. J. Auton. Nerv. Syst. 43, 111. Verdu, ´ E., Navarro, X., 1997. Comparison of immunohistochemical and functional reinnervation of skin and muscle after peripheral nerve injury. Exp. Neurol. 146, 187–196. Verdu, ´ E., Butı, ´ M., Navarro, X., 1995. The effect of aging on efferent nerve fibers regeneration in mice. Brain Res. 696, 76–82. Verdu, ´ E., Butı, ´ M., Navarro, X., 1996. Functional changes of the peripheral nervous system with aging in the mouse. Neurobiol. Aging 17, 73–77. Verdu, F.J., Ceballos, D., Valero, A., ´ E., Vilches, J.J., Rodrıguez, ´ Navarro, X., 1998. Physiological and immunohistochemical characterization of cisplatin-induced neuropathy in mice, Muscle Nerve Žin press.. Vilches, J.J., Navarro, X., Verdu, ´ E., 1995. Functional sudomotor responses to cholinergic agonists and antagonists in the mouse. J. Auton. Nerv. Syst. 55, 105–111.