Water content, vasoactive intestinal polypeptide and substance P in intact and crushed sciatic nerves of normal and streptozotocin-diabetic rats

Journal of the Neurological Sctences, 1988, 83 167-177

167

Elsevier

JNS 02939

Water content, vasoactive intestinal polypeptide and substance P in intact and crushed sciatic nerves of normal and streptozotocin-diabetic rats P. A n a n d 1, J . G . Llewelyn 2, P. K. T h o m a s 2, K. R. W. Gillon 3,*, R. Lisk 2 and S.R. Bloom 1 XDepartment of Medtcine, Royal Postgraduate Medical School, London (U K ), 2Department of NeurologTcal Science, Royal Free Hospital School of Medicine, London (1.IK ) and 3Department of Biochemistry, Untverstty of Nottingham Medtcal School. Nottingham (U.K_) (Received 21 Apnl, 1987) (Rexased, received 8 September, 1987) (Accepted 10 September, 1987)

S UMMARY

Observations on streptozotocin-diabetic rats have confirmed overhydration of peripheral nerve As in previous studies, the sorbitol and fructose content, when expressed m terms of wet weight of nerve was found to be increased and myo-inosltol decreased. The reduction in myo-inositol content was less, although still significant, when expressed in terms of protein content. Nerve water content increased during WaUerian degenerataon following a crush injury in both normal and diabetic animals, but was relatively less in the latter. Vasoactive intestinal polypepUde (VIP) concentrations were significantly increased in diabetic nerve, those for substance P being normal. Both became severely reduced during Wallerian degeneration following nerve crush and hgature. The significance of these findings is discussed. The accumulation of water in the endoneurial compartment may be related to impaired extraction by the perineunum, to which the increased VIP content may contribute. These changes are unlikely to be responsible for nerve fibre damage.

*Present address Astra Chnical Research Unit, 10 York Place, Edinburgh, U.K Correspondence to- Prof_ P K_ Thomas, Department of Neurologmal Science, Royal Free Hospital School of Medlone, Rowland Hill Street, London NW3 2PF, U K 0022-510X/88/$03 50 © 1988 Elsevier Science Pubhshers B V. (Biomedical DIvision)

168 Key words

Experimental diabetes; Nerve oedema, Nerve injury; Vasoactlve intestinal polypeptlde; Substance P

INTRODUCTION

The mtrafascicular compartment of peripheral nerve trunks possesses spaces that contain endoneurial fluid These are particularly evident immediately internal to the perineurium, around blood vessels and in clefts between small bundles of nerve fibres (Thomas and Olsson 1984). Weiss et al. (1945) and MeUick and Cavanagh (1967), from observations employing tracer substances, demonstrated a proximodlstal flow of endoneurial fluid. Several factors are probably responsible for this, including a progressive increase in size of the endoneurial spaces distally (Stevens et al. 1973). It has been suggested on morphological grounds (Ross and Reith 1959) that the perlneunum possesses contractile properties, raising the posslbihty of peristalac activity, although attempts to demonstrate perineurial contractibility have failed (Thomas and Olsson 1975, Low et al. 1980). The component fascicles in peripheral nerve trunks are bounded by the perineunum which provides a diffusion barrier that isolates the endoneunal compartment. This compartment is "open-ended" m the periphery at neuromuscular junctions and at unencapsulated sensory receptors (see Thomas and Olsson 1984). Peripheral nerves do not possess lymphatic vessels within the endoneurial compartment (Defrise 1930) and fired that has left endoneurial capillaries and has not escaped at the periphery is possibly actively removed by the perineurium (Krnjevlc 1954), along with the extrusion of sodium. The permeurium is known to possess (Na + + K + )-ATPase activity (Shanthaveerappa and Bourne 1962; Llewelyn and Thomas 1987). Endoneunal fluid is hypertonlc with respect to plasma (Krnjevic 1954) and under a shght positive pressure (Low et al. 1977; Myers et al. 1978), in contrast to the negative pressures obtained from interstmal fired in most tissues (Guyton et al. 1971). Endoneurial oedema is a feature of a number of human and experimental neuropathles. These have been reviewed by Low (1984), who has discussed the different mechanisms that may be operative Previous studies indicate that endoneurial oedema probably develops m streptozotocminduced diabetes in rats (Jakobsen 1978; Tomlinson et al. 1986), although an increased water content in nerve was not detected by Gabbay (1973). As pointed out by Tomhnson et al. (1986) the data by Yue et al. (1984) permit the reader to calculate that nerve water content was increased in this study. Endoneurial oedema is a feature in Wallerian degeneration. It is an immediate occurrence m the region of a crush injury (see Olsson 1984), where it is related to local vascular damage. There is a delayed increase in water content distal to the site of injury (Johnson et al 1950) This is associated wath an increase in endoneurial pressure that reaches a peak of 4-5 ames the normal value at 6 days after injury (Powell et al. 1979a), returning to normal after 3 weeks. It is accompanied by a diffuse increase in vascular permeabihty throughout the nerve distal to the lesion (Melhck and Cavanagh 1968)

169 In this study we have reinvestigated the water content of the sciatic nerve in streptozotocin-diabetic rats and have conftrmed an increase. We have examined the changes that occur distal to a crush lesion during Wallerian degeneration in comparison with those in control animals. As vasoactive intestinal polypeplade (VIP) is a strong potentlator of oedema (WiUiams 1982), and as substance P is implicated m the regulation of vascular responses to injury (Burnstock 1977; Hagermark et al. 1978), we have also investigated the concentration of these neuropeptides in diabetic nerve and the alterations that follow nerve injury.

MATERIALS AND METHODS Induction of diabetes Diabetes was reduced in mature male Wistar rats (weight range 280-320 g) by the intravenous injectmn via a tail veto of a buffered solution of streptozotocm (65 mg/kg body weight). Age-matched control rats were admimstered an eqmvalent quantity of physiological saline (150 mmol/1). Diabetic and control animals were maintained separately m plastic metabolic cages and fed on 41B Oxoid diet (Oxoid, Basingstoke, Hants, U.K.) with unlmaited water. The 41B Oxold diet was found to contain 0.023~o inositol. Both groups of animals were weighed regularly. Diabetic rats developed glycosuna m excess of 2 ~ within a few days of administration of streptozotocin and progressively lost weight. For the 3 experiments performed (see below) both groups were maintained for a period of 2 months. When anaesthetized at the termination of the experiment, a blood sample was taken by intracardiac aspiration and plasma glucose determined on an Encore Chemistry System analyzer (Baker Instruments Corporation, Allentown, PA, U.S.A.). Productton of nerve crush lesions Under general anaesthesia with intramuscular Hypnorm (Janssen, Crown Chemicals, Lamberhurst, Kent, U.K.) and halothane (ICI, Macclesfield, Cheshire, U.K.) and oxygen inhalation, the left sciatic nerve was exposed in the thagh. It was fLrmly crushed for 10 s m the upper thigh with smooth-tipped watchmakers' forceps. Previous observations in our laboratory have established that tlus interrupts all myehnated and unmyelinated axons in the nerve. In Experiment 2, the site of crush was marked by a black silk suture stitched into the adjacent muscle. The crush lesions in Experiment 3 were marked by a black silk suture tied firmly around the nerve at the site of the crush to impede regeneration. The wound was closed and the animals allowed to recover from the anaesthetic. Sciatic nerve sampling Experiment 1 This was performed on ammals not subjected to nerve crush lesions. Under general anaesthesia as above, a 3-era length was removed from the left sciatic nerve for the estimation of wet and dry weights. For the former it was weighed immediately after removal and later dned at 80 °C until there was no further change in

170 weight and the dry weight recorded. The water content was expressed as a percentage of wet weight. The opposite nerve was removed, frozen in liquid nitrogen and stored at - 7 0 °C for the later estimation of glucose, fructose, myo-inositol and sorbltol as described below.

Experiment 2. In the second experiment, 6 days after crush injury, the sciatic nerve was removed and a segment 1 cm m length taken 0.6 cm below the site of the crush for the measurement of wet and dry weights and the estimaUon of water content. A simalar segment was removed from the opposite uncrushed scmtic nerve Experiment 3. In a third experiment, 6 days following nerve crush, under general anaesthesia as above, the left sciatic nerve was excised and two segments, each 0 6 cm in length, were taken distal to the crush site. Similar segments were obtained from the uncrushed nerve on the right. This tissue was Lmmediately processed for VIP and substance P estamatlon as described below. Biochemzcal methods Perchlonc acid extracts of weighed frozen sciatic nerve samples were prepared as described by Palmano et al. (1977) for the analysis of free mosltol. Portions of the HC104 supernatant were neutralized with saturated K H C O 3 solution, deionized by passage through Amberlite MB3 mixed-bed ion exchange resin (British Drug Houses), and freeze-dried in preparation for gas-hquld chromatographic (GLC) analysis. Inositol and free sugars m freeze-dried lassue extracts and hydrolysates were converted into then" tnmethylsilyl ethers (Sweeley et al. 1963) by reaction with 100 #1 of pyridine/hexamethyldisilazane/-tnmethylchlorosilane (5 : 2 1, by vol.). The tubes were shaken mechanically for 4 h to ensure complete reaction. Samples were chromatographed on a Pye series 104 gas-liqmd chromatograph equipped with hydrogen flameionization detectors. Portions (1-2/zl) of the reactaon mixture were injected with a 10 #1 microsyrmge on to a 180-cm glass column packed with 3 ~ SE 30 on Supelcoport (Supelco, Belleforte, PA, USA) and chromatographed with N 2 as carrier gas (flow rate 35 ml/rmn) and an oven temperature programme of 180 °C for 6 mm followed by an increase of 3 ° C/mm to 200 ° C. Sugars were identified by comparison of retention times with those of known standards, and quantificaUon was effected by relating peak area of sugar (peak height x width at 1/2 peak height) to that of an internal standard (methyl-at-D-mannopyranoside) added to samples before lyophihsatlon. Assay of VIP and substance P Each sciatic nerve sample was weighed, its length measured and its peptide content extracted by boiling m 0.5 M acetic acid (1.10, w/v) for 10 mm. The extracts were stored at - 2 0 °C before assay Duphcate 10-#1 ahquots of each extract were assayed for VIP and substance P using prewously described sensltwe and specific radioimmunoassays (Mitchell and Bloom 1983; McGregor and Bloom 1983). Following 5 days incubation with iodinated tracers, "bound" and "free" fractions were separated using an actwated charcoal method and counted in a gamma counter. The

171 molecular forms of the immunoreactivities were characterized by S e p h a d e x gel filtration. Peptide immunoreactivities were eluted with Kav values similar to those o f their respective pure peptide s t a n d a r d s (VIP expected G - 5 0 Kay 0.55, o b s e r v e d 0.52; substance P expected G-25 Kay 0.33; o b s e r v e d 0.35) Statistical analysis

The results are expressed as m e a n s + S E M . Statistical analysis was p e r f o r m e d using the unpaired S t u d e n t ' s t-test.

RESULTS The values for b o d y weight a n d p l a s m a glucose concentrations in the first two experiments are shown in Table 1. The diabetic animals h a d a considerably r e d u c e d b o d y weight a n d significant hyperglycaemia (P < 0.001) when c o m p a r e d with the age-matched controls. T h e rats in Experiment 1 were m a d e diabetic at a slightly greater age a n d were less severely hyperglycaemic. Sciatic nerve water content

T h e nerves from diabetic animals were found to be o v e r h y d r a t e d as c o m p a r e d with the control animals. The results are given in Table 1. F o r Experiment 1, water content was 71.4% o f wet weight o f nerve in the diabetic rats as c o m p a r e d with 64.29/0 m the controls. The c o r r e s p o n d i n g values for Experiment 2 were 70.2~o and 60.3% respectively. The differences are b o t h significant (P < 0.05 for Experiment 1, P < 0.01 TABLE 1 BODY WEIGHT, PLASMA GLUCOSE AND SCIATIC NERVE WATER CONTENT FOR CONTROL AND DIABETIC RATS Values are expressed as mean + SEM Experiment 1~

Experiment 2b

580.6 + 9.9 + 42 2 + 153+ 64 2

12.2

426 7 + 18 1

07

11 0 _+ 0 5

23 12

12 1 _+ 0 6 4 8 + 0.3 60 3

Controls

Weight (g) Plasma glucose (mmol/1) Nerve wet weight (mg) Nerve drywelght(mg) Nerve water content (% wet weight) Dtabet~c

Weight (g) Plasma glucose (retool/l) Nerve wet weight (mg) Nerve dry weight (mg) Nerve water content (% wet weight) Controls, n = 10, diabetic, n = 8_ b Controls, n = 10, diabetic, n = 9

439.9 + 9.7 28.4 + 0 8 48 7 + 2.3 13 6 + 0.7 71 4

223 3 + 7 4 45 4 + 2 1

11 4 + 0 8 34 + 02 70 2

172 TABLE 2 EFFECT OF SCIATIC NERVE CRUSH ON WET WEIGHT, DRY W E I G H T AND PERCENTAGE WATER CONTENT IN DIABETIC AND AGE MATCHED CONTROL RATS Values are expressed as mean + SEM Number of observations is shown in parentheses

Side of crush

Wet wt (mg) Contra

Control Dmbetlc

Dry wt (mg) Ips~

121+_06(8) 182+-05(8) P < 00001 114+-08(9) 1 3 9 + 1 I (9) P < 0.05

Contra

~o~water content Ipsl

4_8 + 0.3 (8) 56+-04(8) P<005 34+02(8) 35+-02(8) ns

Contra

Ipsl

603

692

702

748

Contra = contralateral to crush, Ipsl = lpsdateral to crush, n.s = not slgmficant

for Experiment 2) but slightly greater for the more severely diabetic animals of Expertment 2. In Experiment 2 (see Table 2), following crush injury there was a 50.4~o increase in the wet weight of the nerve distal to the crush in the control animals (P < 0.0001). The diabetic nerves, in contrast, showed only a 21.9Yo increase m wet weight following injury (P < 0 05) Table 1 shows that the mean dry weight of nerve was less m the diabetic animals as compared with the controls m both Experiments 1 and 2. This presumably reflects reduced growth in the diabetic animals over the two month period after the induction of diabetes (see Sharma et al. 1981).

Sciattc nerve glucose, fructose, sorbitol and myo-inositol content The results of the biochemical estimations on the sciatic nerve samples obtained in Experiment 1 are shown m Table 3. This demonstrates that the concentrations of TABLE 3 SCIATIC NERVE GLUCOSE, FRUCTOSE, SORBITOL AND MYO-INOSITOL IN CONTROL AND DIABETIC RATS Values (In #mol/g wet wt) are expressed as means + SEM The number of observations is shown in parentheses

Control Diabetic P

Glucose

Fructose

Sorbltol

Myo-mositol

2 50 + 0 26 (10) 841 + 0 40 (10) < 0 001

0.83 + 0 07 (10) 6 25 + 0 31 (10) < 0 001

0 16 (1) 2_29 + 0 21 (10)

3 25 _+ 0 22 (10) 1 69 + 0 05 (10) < 0.001

0_013 + 0_001 (10) 0 160 + 0 017 (10) <0001

0 003 (1) 0.058 + 0 006 (10)

0 050 + 0_002 (10) 0 043 + 0 003 (10) <005

Values expressed as gmol/mg protein" Contol Diabetic P

0 038 + 0 002 (10) 0_213_+ 0_017 (10) <0001

173 glucose and fructose per wet wt o f nerve were significantly increased (P < 0.001) in the diabetic as c o m p a r e d with the control nerves. Sorbitol could only be quantltated m 1 control nerve m which it was 0.16/zmol/g wet wt; in the other control nerves, the concentration was therefore less than this value. In a previous study (Jefferys et al. 1978) we obtmned a value of 0.08 + 0.05 (SD) #mol/g wet wt. nerve for normal rats. The value of 2.29 + 0.21 ( S E M ) / z m o l / g wet wt. in the diabetic animals is therefore clearly increased. The myo-inositol concentration was reduced in the diabetic nerves at 1.69 + 0.05/zmol/g wet wt as compared with 3.25 + 0.22 #mol/g wet wt in the controls (P < 0.001). These findings are comparable to those obtained in other studies (e.g., Greene et al. 1975). The results have also been expressed in terms of nerve protein content. The glucose and fructose concentrations were again both significantly increased (P < 0.001) and the sorbltol concentration was 0.058 + 0.006 # m o l / m g protein as compared with 0.003 # m o l / m g protein for the single control nerve. The reduction was less for myo-mosltol when expressed in terms o f protein content, being 0.043 + 0.003 #mol/g protein m the diabetic antmals as compared with 0.050 + 0.002 #mol/g protein in the controls, but was still significant (P < 0.05).

VIP and substance P assays VIP and substance P were assayed in the right sciatic nerve from streptozotocindiabetic rats and c o m p a r e d with the values for control animals. The results are given in Table 4. It will be seen that there is a highly significant increase in VIP but no significant difference for substance P. Neuropeptide contents calculated per unit length of nerve showed no significant difference from the above results. In nerves that had been crushed and a ligature tied around the site o f the crush to impede regeneration, no significant amounts of VIP or substance P were detectable distal to the lesion at 6 days postoperation. These observations were made on 8 diabetic and 8 control animals.

TABLE 4 NEUROPEPTIDE CONTENT OF INTACT AND CRUSHED SCIATIC NERVE FROM CONTROL AND DIABETIC RATS Values expressed as pmol/g wet weight (mean + SEM)

Intact nerve VIP Substance P

Control

Diabetic

P

2.28 + 0 18 (n = 18) 12.06 + 0_55 (n = 10)

5 16 + 029 (n = 20) 13 01 + 1 01 (n = 10)

n_s

Crushed nerve (distal to hgation) VIP < 0_8 (n = 8) Substance P <05 (n = 8)

< 0.8 (n = 8)

<05 (n = 8)

<0 005

174 DISCUSSION These results have confirmed that diabetic nerve is overhydrated. From the hlstometric fmdings obtained by Jakobsen (1978), it can be 9oncluded that the water accumulates in the endoneurial spaces, which he found were expanded. The accumulation is not within axons, the calibre of which is slightly less than in control animals (Jakobsen 1976) and it is not wlthm Schwann cell cytoplasm which is reduced in cross-sectional area (Jakobsen 1978). The failure to fred an increase in water content m the sciatic nerves of streptozotocm-dlabetic rats by Gabbay (1973) may have been related to dehydration. The explanation for the endoneurial oedema in &abetic nerve is uncertain Vascular permeability was reported to be increased by Seneviratne (1972) but although subsequent studies failed to confirm this observation (Jakobsen et al. 1978; Sima and Robertson 1978; Carson and Hanker 1980), Rechthand (1986) found that the permeability of endoneunal blood vessels to small molecules is increased m streptozotocin diabetic rats. Sorbltol concentrations are increased m diabetic nerve. Recently, Tomlinson et al. (1986) have reported that the aldose reductase inhibitor Statil prevents the accumulatmn of both sorbitol and water in the sciatic nerve of streptozotocindiabetic rats. They therefore suggested that the mcrease in water content is driven osmotically by the increase in sorbitol and fructose. However, the mcrease for both of these substances is in the micromolar range and it ~s thus doubtful if they could be responsible (see Brown and Greene 1984). Possibly the increased flux in the sorbltol pathway has a deleterious effect on the extraction of water from the endoneurial compartment by the perineurium. This is currently under study in our laboratory. The seventy of the endoneurial oedema is relatively modest, and it is unlikely to give rise to nerve fibre damage. The reduction in myo-inositol content in diabetic nerve (Greene et al 1975) was confLrmed, but it is less when expressed in terms of nerve protein content than for wet weight of nerve. Tomhnson et al. (1986) have recently found that the reduction in myo-lnositol content was less when expressed in terms of unit length of nerve than for wet weight, but the disparity was less than obtained m the present study. The VIP content of diabetic nerve was found to be significantly increased as compared with control values. VIP is a strong potentiator of oedema (Williams 1982) and it is therefore possible that it has an additive effect m mcreasmg the water content of diabeUc nerve. The degree of endoneurial oedema is substantmlly greater in galactosaemic neuropathy (Powell et al. 1979b), as is endoneurial fired pressure, in comparison with experimental diabetic neuropathy (Powell et al. 1981) However, we have recently found (personal unpubhshed observations) that the VIP content of nerve is not increased in galactose-fed rats. It is becoming increasingly clear that galactosaemlc neuropathy differs in a number of important respects from dmbetic neuropathy and should therefore not be taken as a model for the changes produced by diabetes. Thus although the nerve accumulates galact~tol and has a reduced content of myo-mositol, endoneurlal (Na ÷ + K÷)-ATPase activity, instead of being reduced, is increased (Lambourne et al. 1987; Llewelyn et al. 1987).

175 No alteration in the content of substance P was observed in the uninjured &abetlc nerves as compared with the values for the control animals. This, in conjunction with the increased VIP concentration, parallels the changes found in entenc nerves of streptozotocin-dlabetac rats. Belai et al. (1985) found a consistent increase in fluorescence intensity for VIP-llke lmmunoreactivity in nerve fibres in the myenteric plexus and circular muscle layer in the ileum and proximal colon without apparent alteration in substance P content assessed lmmunohistochemically. An increase in VIP content was confirmed biochermcally. H o w far such changes in neuropeptldes might contribute to the symptoms of diabetic neuropathy is at present a matter of speculation. It ~s of interest that the percentage increase m water content during Wallenan degeneration, at least when assessed at 6 days when the increase m endoneurial pressure is at its height (Powell et al. 1979a), xs less in diabetic nerves as compared with controls. The explanation for this observation is uncertain. It is not related to any d~fferential changes m the content of substance P or VIP, as both VIP and substance P were barely detectable in the nerve below the lesion at 6 days following nerve crush and ligation. VIP is known to be translocated in the fast anterograde transport system in axons (Lundberg et al. 1981). The present observations in&cate that in the sciatac nerve of the rat, the large proportion of VIP and substance P travels down axons within the nerve trunk. Little appears to enter locally in nerves accompanying the nutnent vessels. Possibly the relative reduction in the increase in water content in diabetic nerve dunng Wallerian degeneration is related to the fact that intrafasclcular pressure is already increased in uninjured diabetic nerve (Powell et al. 1981), as a consequence of increased water content, as is capillary permeability to small molecules (Rechthand 1986). It would thus again be of interest to establish what occurs in galactosaem~c neuropathy in which endoneurlal fluid pressure is more severely increased (Powell et al. 1979b) ACKNOWLEDGEMENTS This work was supported by grants from the National Fund for Research into Crippling Diseases and Pfizer Ltd. J. G. L. was a Pfizer Research Fellow. K. R. W. G. was supported by a grant to Prof. J.N. Hawthorne from the British Diabetic Association. The streptozotocin was kindly supplied by the Drug Development Branch, National Institutes of Health, Bethesda, MD, U.S.A. The blood glucose estimations were performed by the Department of Chermcal Pathology and Metabolic Medicine, Royal Free Hospital School of Medicine. The neuropeptide assays were performed at the Hammersmith Hospital, London, with the assistance of Drs G . P . McGregor and M.A. Blank

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