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Guanethidine-induced adrenergic sympathectomy augments endoneurial perfusion and lowers endoneurial microvascular resistance
112
Brain Research, 519 (1990) 112-117 Elsevier
BRES 15555
Guanethidine-induced adrenergic sympathectomy augments endoneurial perfusion and lowers endoneurial microvascular resistance Douglas W. Zochodne, Zhongxian Huang, Kim K. Ward and Phillip A. Low Neurobiology Laboratory, Mayo Clinic, Rochester, MN 55905 (U.S.A.) (Accepted 28 November 1989) Key words: Sympathectomy; Nerve blood flow; Endoneurial microvascular resistance; Guanethidine sulfate
Chronic administration of guanethidine sulfate in the rat induces a selective adrenergic neuropathy. We studied the effects of guanethidine-induced adrenergic sympathectomy on rat sciatic nerve blood flow (NBF), microvascular resistance (MR), vessel caliber and norepinephrine (NE) content. A control group of animals was studied following chronic administration of mammalian Ringer's solution. NBF and MR were measured with an endoneurial microelectrode, using the technique of hydrogen clearance (HC). Following HC, the sciatic nerve was perfused with India Ink, removed, frozen and sectioned. Measurements were made of endoneurial microvessel numbers, diameter, circumference and area. The contralateral sciatic nerve was removed for measurements of NE content. In guanethidine-treated animals we observed elevated NBF, reduced MR and dilated microvessels. Numbers of microvessels and fascicular areas were similar to controls. NE content was markedly reduced following sympathectomy. These studies suggest that NBF, unlike cerebral blood flow (CBF), is regulated by its adrenergic input. Removal of adrenergic innervation of the vasa nervorum appears to result in a loss of tonic vasoconstrictive action.
INTRODUCTION Microvascular networks are regulated by overlapping systemic and neurogenic mechanisms 17. In peripheral nerve, local autoregulation appears insignificant 13'e°'21. Vasa n e r v o r u m , however, are innervated by adrenergic, serotonergic and peptidergic axons 2's. The distribution of n o r e p i n e p h r i n e (NE) containing terminals, m o r e o v e r , appears selective. Epineurial and perineurial, but not endoneurial vessels, are innervated 16. Previous investigations in our l a b o r a t o r y d e m o n s t r a t e d that non-specific a - a d r e n e r g i c b l o c k a d e by selective infusion of phentolamine into the arterial supply of the sciatic nerve t e m p o r a r i l y elevated endoneurial nerve blood flow ( N B F ) and lowered M R (microvascular resistance), suggesting that tonic adrenergic input, perhaps at the level of the epineurial feeding vessel, regulates flow in this c o m p a r t m e n t z4. These findings imply that N B F differs significantly from C B F (cerebral blood flow) in which autoregulation is powerful but adrenergic control inconsequential 11. In this study, we examined the changes, following adrenergic s y m p a t h e c t o m y , in N B F and M R using h y d r o g e n clearance (HC), in vessel caliber following India Ink perfusion, and in nerve N E content. Chronic administration of guanethidine sulfate to adult rats induces a selective adrenergic s y m p a t h e c t o m y 14. In pre-
vious investigations of guanethidine s y m p a t h e c t o m y , we observed evidence of selective adrenergic n e u r o p a t h y sparing somatic and p a r a s y m p a t h e t i c function, depletion of peripheral nerve N E content and dilatation of microvessels in the rat cervical sympathetic trunk 23"25. Using an identical s y m p a t h e c t o m y protocol in this study, guanethidine sulfate injected animals were c o m p a r e d to control animals injected with m a m m a l i a n Ringer's solution.
MATERIALS AND METHODS Animals All animals were male Sprague-Dawley (Charles River, WI), albino rats weighing 190-240 g. There were no food or water restrictions. Drug administration Guanethidine monosulfate (Ciba-Geigy, Pharmaceutical Division, Suffern, NY) was administered as an aqueous solution adjusted to pH 7.40 in a dose of 48 mg/kg by daily intraperitoneal injection for 5 weeks. Control animals received mammalian Ringer's solution (composition (per litre): Na + 145 mM; K ÷ 3.5 mM; Ca2÷ 2.0 mM; CI 150 mM; glucose 6.0 mM; HEPES buffer 5 mM; pH 7.40). Protocol At the onset of the experiment, 20 animals were randomly assigned to each of the control and drug treatment groups. Animals were weighed, then anaesthetized for initial nerve conduction studies of the left sciatic nerve. The technique employed for nerve conduction studies has been previously reported23. Injections were begun following recovery from pentobarbital (65 mg/kg).
Correspondence: P.A. Low, Neurophysiology Laboratory, 8th Floor Guggenheim, Mayo Clinic, Rochester, MN 55905, U.S.A. 0006-8993/90/$03.50 (~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)
113 The final injection was given one day prior to the final studies. Rats were reweighed, anaesthetized with pentobarbitai (65 mg/kg) or Inactin (100 mg/kg), had repeat left sciatic nerve conduction studies, then underwent a midline ventral neck incision for tracheostomy and placement of a left carotid intra-arterial catheter (PE-50; Intramedic). The mean systemic arterial blood pressure (MAP) was measured through the carotid line using a pressure transducer (Statham 23 dB, Hato Rey, PR) and polygraph chart recorder (Model 7D Polygraph, Grass Instruments, Quincy, MA). The rats were then paralyzed using D-tubocurarine (1 unit in the arterial line and 2 units intraperitoneally) and ventilated (Harvard Rodent Respirator, South Natick, MA). Arterial blood gases were measured every 30-60 rain (BMS3Mk2 Blood Micro System; Radiometer, Westlake, OH) to ensure the values remained within the physiological range. Each animal then underwent HC measurements of the left sciatic nerve, removal of the right sciatic nerve for NE measurements followed by India Ink perfusion of the lower limbs.
TABLE II
N B F and M R measurements The technique of NBF measurement using HC in our laboratory has been previously reported 13. Each animal had two HC measurements with the microelectrode placed within the mid-portion (above its trifurcation) of the left sciatic nerve. In addition to polygraph recording, signal during HC was also acquired by computer (IBM PCXT; Labtech Notebook data acquisition software, Wilmington, MA) using an A/D converter (Coulbourn L25-08, Lehigh Valley, PA). Acquired data were curve-fitted to a mono- or bi-exponential curve (Labtech Notebook, Boston, MA) for calculation of NBF values. MR was calculated as MAP/NBE
ments were limited to the luminal area perfused with India Ink. Oblong vessel profiles had mean vessel diameter calculated as the diameter derived from area measurement, assuming circularity. Extrafascicular leakage of India Ink prevented accurate measurements of epineurial vessels.
Morphologic studies Following HC, animals underwent catheterization of the right iliac artery (PE-90; Intramedic) and were perfused with India Ink suspended and diluted in mammalian Ringer's solution and heparin for 30 min (approximate total volume infused was 30-40 ml per rat at an infusion pressure of 100-125 mm Hg). Upon completion of perfusion, the left sciatic nerve was removed and placed immediately in a freezer (-20 or -70 °C). Specimens were then later mounted on a chuck using isopentane and liquid nitrogen and sectioned (10/zm thick) at a level 2 mm distal to the sciatic notch. At this level the sciatic nerve is monofascicular. Measurements were made of fascicular area and endoneurial microvessel numbers, area, perimeter and diameter using a microscope (Nikon Optiphot, Garden City, NY) interfaced with a computerized image analysis system (MCID, St. Catherine's, Ontario) of three technically satisfactory sections at the same level (final magnification x 170). A mean value per animal was obtained of each animal and compared between the two groups. All measurements were conducted without knowledge of the group category of the slide. Microvessel measure-
Statistics Statistical calculations were conducted on each of 3 separate programs: a Hewlett-Packard HP 9845T computer with Data Manipulation and General Statistics programs; an IBM PS/2 computer (Epistat); a Digital computer (Clinfo program). Twotailed tests were used to compare groups. Non-parametric data were compared using the Mann-Whitney test of rank sums. Null hypotheses were rejected when P < 0.05.
Results o f NB F and N E measurements
Results are in mean + S.E.M. Significance refers to P-value calculated using a 2-tailed Student's t-test. Parameter
NBF (ml/100 g/min) MR (mm Hg/ml/ 100g/min) MAP (mm Hg) NE (ng/mg wet wt.)
Guanethidine (n = 20)
Control (n = 20)
19.9 + 1.5
14.8 + 0.7
7.18 -+ 0.47 130.8 _+2.6 0.04 + 0.01 (n = 11)
10.84 + 0.59 152.8 + 3.1 0.79 + 0.11 (n = 10)
Significance
0.003 <0.001 <0.001 <0.001
Norepinephrine measurements Non-desheathed nerves were weighed and NE content determined using the tissue preparation method of Chad et al. 5 and high-pressure liquid chromatography with electrochemical detection 1°.
RESULTS
General observations and nerve conduction
As previously observed23, guanethidine-treated animals gained less weight than controls and developed
TABLE III Morphometric studies
TABLE I
Results are in mean + S.E.M. Significance refers to P-value obtained using a 2-tailed Student's t-test.
Weights and conduction studies
Results are mean + S.E.M. Significance refers to P-value calculated using a 2-tailed Student's t-test.
Parameter
Parameter
Sampled area (a mz × 105) Microvessel numbers Total microvessel lumen area (a m2 × 103) Mean microvessel lumen area (am z x 102) Mean microvessel perimeter (am)
Guanethidine (n = 20)
Onset wt. (g) 221.7 + 4.5 Final wt. (g) 315.4 -+ 4.4 Sciatic-tibial CV (m/s) Onset 39.9 _+ 1.1 Final 52.2 + 1.8 Amplitude (mV) Onset 3.84 + 0.33 Final 5.92 + 0.33
Controls (n = 20)
Significance
222.8 + 4.0 365.5 -+ 5.6
NS <10 6
38.5 _+ 1.2 50.7 _+ 1.9
NS NS
4.19 + 0.23 5.38 -+ 0.38
NS NS
Guanethidine (n = 12)
Controls (n = 12)
Significance
6.37 + 0.27 18.5 + 1.4
6.41 _ 0.37 18.3 _+ 2.1
NS NS
5.48 _+ 0.66
2.14 + 0.22
<0.001
3.19_+4.4
1.25_+ 1.4
0.001
54.7 + 3.9
31.7 + 2.7
<0.001
114
Fig. 1. Appearance of endoneurial microvessels following India Ink perfusion in sham-injected control animals (A) and guanethidine-treated animals (B). The India Ink stains the lumen of endoneurial vessels (open arrow) and the perineurium (arrow).
ptosis. Sciatic-tibial nerve conduction studies did not significantly differ between the control and guanethidinetreated groups both at the onset of the experiments and following 5 weeks of treatment. Conduction velocity and c o m p o u n d motor action potential amplitudes increased over the 5 weeks in each rat as predicted by normal maturational changes ~9. Results are included in Table I.
N B F and M R measurements
At the end of the 5 week treatment period, NBF in the guanethidine-treated animals was 34% greater than that of controls (Table II). Control values were similar to control values previously reported ~3. Results were also compared to a second group of control animals with weights similar to that of the guanethidine group. Both
115 TABLE IV
250[
=_=
2001
= _-
Influence of anaesthesia technique
Results are in mean + S.E.M. Significance refers to P-value calculated using a 2-tailed Student's t-test. C = control animals; G = guanethidine animals.
I~1 control ~] guonethidine
150
Parameter significance
Pentobarbital (n = 6)
lnactin (n = 6) L. 1 0 0
Sampled area (,um2 x 105) C G
6.40 + 0.40 6.36+0.56
6.42 + 0.40 NS 6.37+0.56 NS
Microvessel numbers
13.0+ 1.3 16.2+ 1.4
23.6 + 2.6 20.8 +2.2
50
C G
0.004 NS 0
Total microvessel lumen area (um2 x 103) Mean microvessel lumen area Q~m2 x 102) Mean microvessel perimeter (um) NBF (mi/100 g/min) (n = 10)
C G
2.03 +0.27" 6.49+ 1.18
2.25 + 0.42* NS 4.47+0.31 NS
C G
1.58+ 0.20* 4.03 +0.64
0.92 + 0.09* 0.004 2.36 +0.42 0.053
C G
38.2 + 3.6* 60.9 + 4.8
25.3 + 1.2" 0.007 48.5 + 5.5 NS
C
15.1 +
0.8 19.3+2.2
14.4 + 1.2" 20.5+2.0
NS NS
10.3 + 0.8*
11.4 + 0.9*
7.6 + 0.8
6.8 + 0.6
NS NS
154.6+4.9" 129.1 + 3.2
NS NS
G MR (mm Hg/ml/100 g/min) C (n = 10) G MAP(mmHg)(n=10)
150.9+4.0" G 132.5 + 4.1 C
* Difference between C vs G group significant at 0.05 level using a 2-tailed t-test.
control groups had similar N B F values regardless of weight. The type of anaesthesia (pentobarbital in 10 animals in each group; Inactin in 10 animals in each group) had no significant influence on N B F results in either the control or guanethidine-treated groups (Table IV) although Inactin anaesthesia was deemed preferable because it provided more stable long-term anaesthesia and because Inactin has been used in our previous H C studies. Experimental variability in N B F measurements made during Inactin anaesthesia in guanethidine-treated animals was less than during pentobarbital anaesthesia (Table IV). Within the Inactin subgroup N B F in the guanethidine-treated group was significantly greater than controls. Within the pentobarbital subgroup this trend was also evident, but did not reach statistical significance. M R fell by 34% and M A P by 14% in the guanethidinetreated group (Table II). M R was not significantly influenced by the type of anaesthesia (Table IV). M R was significantly less in guanethidine-treated than in control
5
10
3o 35 4o 45 15 20 25 Capillary diameter in micrometers
'so
55
60
Fig. 2. Histogram of capillary (microvessel) diameters ~m).
animals within each anaesthesia subgroup. M A P was not influenced by the type of anaesthesia and was significantly lower in guanethidine-treated animals within each anaesthesia subgroup (Table IV). Morphologic studies Results of morphometric studies are given in Table III. Guanethidine-treated and control animals had a similar area of sampling, or faseicular area and similar numbers of perfused microvessels were identified in each group for calculation of luminal areas. Total luminal area of endoneurial microvessels, mean microvessel area, mean microvessel diameter, mean microvessel perimeter and the diameter of the largest microvessel in each fascicle were all significantly greater in guanethidine animals than in controls (Table III and Fig. 2). To exclude a possible effect of the type of anaesthesia, each animal group included 6 pentobarbital-anaesthetized animals and 6 Inactin-anaesthetized animals. Morphometric comparisons were also made between control and drug-treated animals in each of the anaesthesia groups. Sampled fascicular area and total capillary area were independent of anaesthesia type. In both the control and guanethidine-treated animals mean microvessel area was consistently larger in pentobarbital-anaesthetized animals than in Inactin-anaesthetized animals but a larger number of microvessels were perfused in the control Inactin group. Mean microvessel perimeter was larger in the control pentobarbital group than the Inactin group. Within each anaesthesia type, microvessels in guanethidine-treated animals had a larger mean perimeter, mean luminal area and total luminal area than of controls (Table IV). Norepinephrine measurements Norepinephrine content was considerably reduced in the guanethidine-treated group (Table II).
116 DISCUSSION These studies indicate that chronic sympathectomy results in elevated nerve blood flow (NBF), a fall in microvascular calculated resistance (MR) and dilated endoneurial microvessels. In the guanethidine-treated animals, the slow component of hydrogen clearance, which likely represents endoneurial flow, was elevated above values observed in the control sham-injected animals. Similarly flow values were higher than those observed in the sciatic nerve of normal rats in studies using hydrogen clearance or the distribution of iodoantipyrine 13'~5'22. The drug-treated animals gained less weight than controls, but studies in our laboratory have not identified significant variations in HC with animal weights between 280 and 400 g. NBF following sympathectomy was similar to values noted following intra-arterial phentolamine 24. Guanethidine-treated animals had a fall in MAP, but a rise in NBF yielding a lower value of calculated MR. Phentolamine induced a similar effect 24. In the sciatic nerve of non-sympathectomized animals, NBF and MAP have a positive linear correlation - - a fall in MAP would be expected to reduce NBF. The findings of dilated India Ink-perfused microvesseis and loss of NE suggest that lower MR was a result of microvessel dilation deprived of NE-mediated vasoconstriction. We have previously identified that adrenergic NE-mediated vasoconstriction exists by observing shutoff of NBF following local intra-arterial NE delivery. Whole nerve NE is likely derived from adrenergic terminals on vasa nervorum. Similar loss of NE was observed in the vagus, tibial, sural, and peroneal nerves and the cervical sympathetic trunk of rats given guanethidine sulfate in a previous study 23. A non-specific toxic effect of guanethidine does not appear to account for the findings. In this study and previous investigations 23, somatic conduction was not influenced by guanethidine or by the accompanying reduction in weight gain. Guanethidine has a selective effect, sparing cholinergic and somatic nerves 9"14. Technical factors did not permit morphometric analysis of epineurial vessels. Epineurial vessels may be the critical sites of adrenergic influence, determining 'downstream' endoneurial perfusion 24. NE terminals have been noted to be confined to epineurial and perineurial vessels16. Our observations of dilated endoneurial microvessels may imply: (i) that these vessels are also directly innervated by the adrenergic system; (ii) that endoneurial microvessels dilate REFERENCES 1 Altura, A.T. and Altura, B.M., Barbiturates and aortic and venous smooth-muscle function, Anesthesiology, 43 (1975) 432-444. 2 Appenzeller, O., Dhital, K.K., Cowen, T. and Burnstock, G.,
under the conditions of increased 'feeding' flow from the epineurium; and (iii) that NE within the lumen of vessels travels 'downstream' from an epineurial origin into endoneurial vessels, Pericytes with contractile function surround some endoneurial capillaries. An endothelial receptor for NE has been postulated 3. Numbers of India Ink-perfused vessels did not differ between the groups likely excluding significant capillary recruitment in elevating NBF 12. Inactin anaesthesia, which is longer-acting and provides a more stable preparation, resulted in greater numbers of perfused microvessels with a smaller mean caliber than animals treated with pentobarbital. The similar fascicular size would tend to exclude a nonspecific effect on section preparation. A statistically significant difference in NBF between the guanethidine and control subgroups anaesthetized with pentobarbital was not observed despite a trend in the same direction as the Inactin group. A less stable level of anaesthesia with shorter-acting pentobarbital may have accounted for more variable NBF and MAP readings in the guanethidine group. MR, however, was lower in guanethidinetreated animals in both anaesthesia subgroups. Mean microvessels area, total capillary area and mean microvessel perimeter was greater in the guanethidine-treated group irrespective of anaesthesia effects. Circulating catecholamine levels may be higher in the pentobarbitalanaesthetized animals because of a more variable depth of anaesthesia. Circulating catecholamines, a direct drug effect 1 or other factors may have influenced microvessel recruitment and caliber to account for the changes observed, but the precise explanation is unclear. The regulatory characteristics of NBF are different from CBF, which is maintained by autoregulation H. Adrenergic innervation of cerebral vessels has been identified, but appears to have little influence on flow 1~. NBF, on the other hand, has negligible autoregulation 13"2°'21but appears to be influenced by the level of adrenergic 'tone', also a feature of other vascular beds4'6'7. There are likely other mechanisms of NBF regulation, but they are largely unexplored. Serotonin appears to obliterate AV shunt NBF TM and peptidergic influences on NBF have not been examined. Acknowledgements. The authors would like to acknowledge the invaluable technical assistance of James Schmelzerand Paula Zollman. Lisa Fregeau provided expert secretarial assistance. Ciba-Geigykindly provided guanethidine monosulfate. The research was supported in part by grants from NINCDS (R01 NS2 2352, NS14302), MDA, Mogg and Mayo Funds. P.A.L. is the recipient of a Jacob Javits Neuroscience Award. D.W.Z. receivedresearch fellowshipsupport from the Medical Research Council of Canada.
The nerves to blood vessels supplying blood to nerves: the innervation of vasa nervorum, Brain Research, 304 (1984) 383-386. 3 Bevan, J.A. and Duckies, S.P., Evidence for a-adrenergic receptor on intimal endothelium, Blood Vessels, 12 (1975) 307-310.
117 4 Bohlen, H.G. and Gorel R.W., Comparison of microvascular pressures and diameters in the innervated and denervated rat intestine, Microvasc. Res., 14 (1977) 251-264. 5 Chad, D., Bradley, W.G., Rasool, C., Good, P.C., Reichlin, S. and Zivin, J., Sympathetic postganglionic unmyelinated axons in the rat peripheral nervous system, Neurology, 33 (1983) 841847. 6 Delius, W.K., Hagbarth, K.E., Hongell, A. and Wallin, B.G., Manoeuvres affecting sympathetic outflow in human muscle nerves, Acta Physiol. Scand., 84 (1972) 82-94. 7 Delius, W., Hagbarth, K.E., Hongell, A. and Wallin, B.G., Manoeuvres affecting sympathetic outflow in human skin nerves, Acta Physiol. Scand., 84 (1972) 177-186. 8 Dhital, K. and Appenzeller, O., In G. Burnstock and S.G. Griffiths (Eds.), Nonadrenergic Innervation of Blood Vessels, Vol. I1, CRC Press, Boca Raton, FL, 1988, pp. 191-211. 9 Heath, J.W. and Burnstock, G., Selectivity of neuronal degeneration produced by chronic guanethidine treatment, J. Neurocytol., 6 (1977) 397-405. 10 Hegstrand, L.R. and Eichelman, B., Determination of rat brain tissue catecholamines using liquid chromatography with electrochemical detection, J. Chromatogr., 222 (1981) 107-111. 11 Heistad, D.D. and Marcus, M.L., Evidence that neural mechanisms do not have important effects on cerebral blood flow, Orc. Res., 42 (1978) 295-302. 12 Honig, C.R., Odoroff, C.L. and Frierson, EL., Capillary recruitment in exercise: rate, extent, uniformity, and relation to blood flow, Am. J. Physiol., 238 (1980) H31-H42. 13 Low, P.A. and Tuck, R.R., Effects of changes of blood pressure, respiratory acidosis and hypoxia on blood flow in the sciatic nerve of the rat, J. Physiol. (Lond.), 347 (1984) 513-524. 14 Johnson, E.M. and Manning, P.T., Guanethidine-induced destruction of sympathetic neurons, Int. Rev. Neurobiol., 25 (1984) 1-37. 15 Myers, R.R., Mizisin, A.P., Powell, H.C. and Lampert, P.W.,
Reduced nerve blood flow in hexachlorophene neuropathy. Relationship to elevated endoneuriai fluid pressure, J. Neuropathol. Exp. Neurol., 41 (1982) 391-399. 16 Rechthand, E., Hervonen, A., Sato, S. and Rapoport, S,I., Distribution of adrenergic innervation of blood vessels in peripheral nerve, Brain Research, 374 (1986) 185-189. 17 Renkin, E.M., Control of microcirculation and blood-tissue exchange. In E.M. Renkin, C.C. Michel (Eds.), Handbook of Physiology, Section 2: The Cardiovascular System, Vol. 14, Microcirculation, Part 2, 1984, pp. 627-687. 18 Saxena, P.R. and Verdouw, P.D., Redistribution of 5-hydroxytryptamine of carotid arterial blood at the expense of arteriovenous anastomotic blood flow, J. Physiol. (Lond.), 332 (1982) 501-520. 19 Schmelzer, J.D. and Low, P.A., Electrophysiological studies of the effect of age on caudal nerve of the rat, Exp. Neurol., 96 (1987) 612-620. 20 Smith, D.R., Kobrine, A.I. and Rizzoli, H.V., Absence of autoregulation in peripheral nerve blood flow, J. Neurol. Sci., 33 (1977) 347-352. 21 Takeuchi, M. and Low, P.A., Dynamic peripheral nerve metabolic and vascular responses to exsanguination, Am. J. Physiol., 253 (1987) E349-E353. 22 Tuck, R.R., Schmelzer, J.D. and Low, P.A., Endoneurial blood flow and oxygen tension in the sciatic nerves of rats with experimental diabetic neuropathy, Brain, 107 (1984) 935-950. 23 Zochodne, D.W., Ward, K.K. and Low, P.A., Guanethidine adrenergic neuropathy: an animal model of selective autonomic neuropathy, Brain Research, 461 (1988) 10-16. 24 Zochodne, D.W. and Low, P.A., Pharmacologic manipulation of nerve blood flow, Soc. Neuroci. Abstr., 14 (1988) 996. 25 Zochodne, D.W., Low, P.A. and Dyck, P.J., Adrenergic sympathectomy ablates unmyelinated fibers in the rat 'preganglionic' cervical sympathetic trunk, Brain Research, 498 (1989) 221-228.
Brain Research, 519 (1990) 112-117 Elsevier
BRES 15555
Guanethidine-induced adrenergic sympathectomy augments endoneurial perfusion and lowers endoneurial microvascular resistance Douglas W. Zochodne, Zhongxian Huang, Kim K. Ward and Phillip A. Low Neurobiology Laboratory, Mayo Clinic, Rochester, MN 55905 (U.S.A.) (Accepted 28 November 1989) Key words: Sympathectomy; Nerve blood flow; Endoneurial microvascular resistance; Guanethidine sulfate
Chronic administration of guanethidine sulfate in the rat induces a selective adrenergic neuropathy. We studied the effects of guanethidine-induced adrenergic sympathectomy on rat sciatic nerve blood flow (NBF), microvascular resistance (MR), vessel caliber and norepinephrine (NE) content. A control group of animals was studied following chronic administration of mammalian Ringer's solution. NBF and MR were measured with an endoneurial microelectrode, using the technique of hydrogen clearance (HC). Following HC, the sciatic nerve was perfused with India Ink, removed, frozen and sectioned. Measurements were made of endoneurial microvessel numbers, diameter, circumference and area. The contralateral sciatic nerve was removed for measurements of NE content. In guanethidine-treated animals we observed elevated NBF, reduced MR and dilated microvessels. Numbers of microvessels and fascicular areas were similar to controls. NE content was markedly reduced following sympathectomy. These studies suggest that NBF, unlike cerebral blood flow (CBF), is regulated by its adrenergic input. Removal of adrenergic innervation of the vasa nervorum appears to result in a loss of tonic vasoconstrictive action.
INTRODUCTION Microvascular networks are regulated by overlapping systemic and neurogenic mechanisms 17. In peripheral nerve, local autoregulation appears insignificant 13'e°'21. Vasa n e r v o r u m , however, are innervated by adrenergic, serotonergic and peptidergic axons 2's. The distribution of n o r e p i n e p h r i n e (NE) containing terminals, m o r e o v e r , appears selective. Epineurial and perineurial, but not endoneurial vessels, are innervated 16. Previous investigations in our l a b o r a t o r y d e m o n s t r a t e d that non-specific a - a d r e n e r g i c b l o c k a d e by selective infusion of phentolamine into the arterial supply of the sciatic nerve t e m p o r a r i l y elevated endoneurial nerve blood flow ( N B F ) and lowered M R (microvascular resistance), suggesting that tonic adrenergic input, perhaps at the level of the epineurial feeding vessel, regulates flow in this c o m p a r t m e n t z4. These findings imply that N B F differs significantly from C B F (cerebral blood flow) in which autoregulation is powerful but adrenergic control inconsequential 11. In this study, we examined the changes, following adrenergic s y m p a t h e c t o m y , in N B F and M R using h y d r o g e n clearance (HC), in vessel caliber following India Ink perfusion, and in nerve N E content. Chronic administration of guanethidine sulfate to adult rats induces a selective adrenergic s y m p a t h e c t o m y 14. In pre-
vious investigations of guanethidine s y m p a t h e c t o m y , we observed evidence of selective adrenergic n e u r o p a t h y sparing somatic and p a r a s y m p a t h e t i c function, depletion of peripheral nerve N E content and dilatation of microvessels in the rat cervical sympathetic trunk 23"25. Using an identical s y m p a t h e c t o m y protocol in this study, guanethidine sulfate injected animals were c o m p a r e d to control animals injected with m a m m a l i a n Ringer's solution.
MATERIALS AND METHODS Animals All animals were male Sprague-Dawley (Charles River, WI), albino rats weighing 190-240 g. There were no food or water restrictions. Drug administration Guanethidine monosulfate (Ciba-Geigy, Pharmaceutical Division, Suffern, NY) was administered as an aqueous solution adjusted to pH 7.40 in a dose of 48 mg/kg by daily intraperitoneal injection for 5 weeks. Control animals received mammalian Ringer's solution (composition (per litre): Na + 145 mM; K ÷ 3.5 mM; Ca2÷ 2.0 mM; CI 150 mM; glucose 6.0 mM; HEPES buffer 5 mM; pH 7.40). Protocol At the onset of the experiment, 20 animals were randomly assigned to each of the control and drug treatment groups. Animals were weighed, then anaesthetized for initial nerve conduction studies of the left sciatic nerve. The technique employed for nerve conduction studies has been previously reported23. Injections were begun following recovery from pentobarbital (65 mg/kg).
Correspondence: P.A. Low, Neurophysiology Laboratory, 8th Floor Guggenheim, Mayo Clinic, Rochester, MN 55905, U.S.A. 0006-8993/90/$03.50 (~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)
113 The final injection was given one day prior to the final studies. Rats were reweighed, anaesthetized with pentobarbitai (65 mg/kg) or Inactin (100 mg/kg), had repeat left sciatic nerve conduction studies, then underwent a midline ventral neck incision for tracheostomy and placement of a left carotid intra-arterial catheter (PE-50; Intramedic). The mean systemic arterial blood pressure (MAP) was measured through the carotid line using a pressure transducer (Statham 23 dB, Hato Rey, PR) and polygraph chart recorder (Model 7D Polygraph, Grass Instruments, Quincy, MA). The rats were then paralyzed using D-tubocurarine (1 unit in the arterial line and 2 units intraperitoneally) and ventilated (Harvard Rodent Respirator, South Natick, MA). Arterial blood gases were measured every 30-60 rain (BMS3Mk2 Blood Micro System; Radiometer, Westlake, OH) to ensure the values remained within the physiological range. Each animal then underwent HC measurements of the left sciatic nerve, removal of the right sciatic nerve for NE measurements followed by India Ink perfusion of the lower limbs.
TABLE II
N B F and M R measurements The technique of NBF measurement using HC in our laboratory has been previously reported 13. Each animal had two HC measurements with the microelectrode placed within the mid-portion (above its trifurcation) of the left sciatic nerve. In addition to polygraph recording, signal during HC was also acquired by computer (IBM PCXT; Labtech Notebook data acquisition software, Wilmington, MA) using an A/D converter (Coulbourn L25-08, Lehigh Valley, PA). Acquired data were curve-fitted to a mono- or bi-exponential curve (Labtech Notebook, Boston, MA) for calculation of NBF values. MR was calculated as MAP/NBE
ments were limited to the luminal area perfused with India Ink. Oblong vessel profiles had mean vessel diameter calculated as the diameter derived from area measurement, assuming circularity. Extrafascicular leakage of India Ink prevented accurate measurements of epineurial vessels.
Morphologic studies Following HC, animals underwent catheterization of the right iliac artery (PE-90; Intramedic) and were perfused with India Ink suspended and diluted in mammalian Ringer's solution and heparin for 30 min (approximate total volume infused was 30-40 ml per rat at an infusion pressure of 100-125 mm Hg). Upon completion of perfusion, the left sciatic nerve was removed and placed immediately in a freezer (-20 or -70 °C). Specimens were then later mounted on a chuck using isopentane and liquid nitrogen and sectioned (10/zm thick) at a level 2 mm distal to the sciatic notch. At this level the sciatic nerve is monofascicular. Measurements were made of fascicular area and endoneurial microvessel numbers, area, perimeter and diameter using a microscope (Nikon Optiphot, Garden City, NY) interfaced with a computerized image analysis system (MCID, St. Catherine's, Ontario) of three technically satisfactory sections at the same level (final magnification x 170). A mean value per animal was obtained of each animal and compared between the two groups. All measurements were conducted without knowledge of the group category of the slide. Microvessel measure-
Statistics Statistical calculations were conducted on each of 3 separate programs: a Hewlett-Packard HP 9845T computer with Data Manipulation and General Statistics programs; an IBM PS/2 computer (Epistat); a Digital computer (Clinfo program). Twotailed tests were used to compare groups. Non-parametric data were compared using the Mann-Whitney test of rank sums. Null hypotheses were rejected when P < 0.05.
Results o f NB F and N E measurements
Results are in mean + S.E.M. Significance refers to P-value calculated using a 2-tailed Student's t-test. Parameter
NBF (ml/100 g/min) MR (mm Hg/ml/ 100g/min) MAP (mm Hg) NE (ng/mg wet wt.)
Guanethidine (n = 20)
Control (n = 20)
19.9 + 1.5
14.8 + 0.7
7.18 -+ 0.47 130.8 _+2.6 0.04 + 0.01 (n = 11)
10.84 + 0.59 152.8 + 3.1 0.79 + 0.11 (n = 10)
Significance
0.003 <0.001 <0.001 <0.001
Norepinephrine measurements Non-desheathed nerves were weighed and NE content determined using the tissue preparation method of Chad et al. 5 and high-pressure liquid chromatography with electrochemical detection 1°.
RESULTS
General observations and nerve conduction
As previously observed23, guanethidine-treated animals gained less weight than controls and developed
TABLE III Morphometric studies
TABLE I
Results are in mean + S.E.M. Significance refers to P-value obtained using a 2-tailed Student's t-test.
Weights and conduction studies
Results are mean + S.E.M. Significance refers to P-value calculated using a 2-tailed Student's t-test.
Parameter
Parameter
Sampled area (a mz × 105) Microvessel numbers Total microvessel lumen area (a m2 × 103) Mean microvessel lumen area (am z x 102) Mean microvessel perimeter (am)
Guanethidine (n = 20)
Onset wt. (g) 221.7 + 4.5 Final wt. (g) 315.4 -+ 4.4 Sciatic-tibial CV (m/s) Onset 39.9 _+ 1.1 Final 52.2 + 1.8 Amplitude (mV) Onset 3.84 + 0.33 Final 5.92 + 0.33
Controls (n = 20)
Significance
222.8 + 4.0 365.5 -+ 5.6
NS <10 6
38.5 _+ 1.2 50.7 _+ 1.9
NS NS
4.19 + 0.23 5.38 -+ 0.38
NS NS
Guanethidine (n = 12)
Controls (n = 12)
Significance
6.37 + 0.27 18.5 + 1.4
6.41 _ 0.37 18.3 _+ 2.1
NS NS
5.48 _+ 0.66
2.14 + 0.22
<0.001
3.19_+4.4
1.25_+ 1.4
0.001
54.7 + 3.9
31.7 + 2.7
<0.001
114
Fig. 1. Appearance of endoneurial microvessels following India Ink perfusion in sham-injected control animals (A) and guanethidine-treated animals (B). The India Ink stains the lumen of endoneurial vessels (open arrow) and the perineurium (arrow).
ptosis. Sciatic-tibial nerve conduction studies did not significantly differ between the control and guanethidinetreated groups both at the onset of the experiments and following 5 weeks of treatment. Conduction velocity and c o m p o u n d motor action potential amplitudes increased over the 5 weeks in each rat as predicted by normal maturational changes ~9. Results are included in Table I.
N B F and M R measurements
At the end of the 5 week treatment period, NBF in the guanethidine-treated animals was 34% greater than that of controls (Table II). Control values were similar to control values previously reported ~3. Results were also compared to a second group of control animals with weights similar to that of the guanethidine group. Both
115 TABLE IV
250[
=_=
2001
= _-
Influence of anaesthesia technique
Results are in mean + S.E.M. Significance refers to P-value calculated using a 2-tailed Student's t-test. C = control animals; G = guanethidine animals.
I~1 control ~] guonethidine
150
Parameter significance
Pentobarbital (n = 6)
lnactin (n = 6) L. 1 0 0
Sampled area (,um2 x 105) C G
6.40 + 0.40 6.36+0.56
6.42 + 0.40 NS 6.37+0.56 NS
Microvessel numbers
13.0+ 1.3 16.2+ 1.4
23.6 + 2.6 20.8 +2.2
50
C G
0.004 NS 0
Total microvessel lumen area (um2 x 103) Mean microvessel lumen area Q~m2 x 102) Mean microvessel perimeter (um) NBF (mi/100 g/min) (n = 10)
C G
2.03 +0.27" 6.49+ 1.18
2.25 + 0.42* NS 4.47+0.31 NS
C G
1.58+ 0.20* 4.03 +0.64
0.92 + 0.09* 0.004 2.36 +0.42 0.053
C G
38.2 + 3.6* 60.9 + 4.8
25.3 + 1.2" 0.007 48.5 + 5.5 NS
C
15.1 +
0.8 19.3+2.2
14.4 + 1.2" 20.5+2.0
NS NS
10.3 + 0.8*
11.4 + 0.9*
7.6 + 0.8
6.8 + 0.6
NS NS
154.6+4.9" 129.1 + 3.2
NS NS
G MR (mm Hg/ml/100 g/min) C (n = 10) G MAP(mmHg)(n=10)
150.9+4.0" G 132.5 + 4.1 C
* Difference between C vs G group significant at 0.05 level using a 2-tailed t-test.
control groups had similar N B F values regardless of weight. The type of anaesthesia (pentobarbital in 10 animals in each group; Inactin in 10 animals in each group) had no significant influence on N B F results in either the control or guanethidine-treated groups (Table IV) although Inactin anaesthesia was deemed preferable because it provided more stable long-term anaesthesia and because Inactin has been used in our previous H C studies. Experimental variability in N B F measurements made during Inactin anaesthesia in guanethidine-treated animals was less than during pentobarbital anaesthesia (Table IV). Within the Inactin subgroup N B F in the guanethidine-treated group was significantly greater than controls. Within the pentobarbital subgroup this trend was also evident, but did not reach statistical significance. M R fell by 34% and M A P by 14% in the guanethidinetreated group (Table II). M R was not significantly influenced by the type of anaesthesia (Table IV). M R was significantly less in guanethidine-treated than in control
5
10
3o 35 4o 45 15 20 25 Capillary diameter in micrometers
'so
55
60
Fig. 2. Histogram of capillary (microvessel) diameters ~m).
animals within each anaesthesia subgroup. M A P was not influenced by the type of anaesthesia and was significantly lower in guanethidine-treated animals within each anaesthesia subgroup (Table IV). Morphologic studies Results of morphometric studies are given in Table III. Guanethidine-treated and control animals had a similar area of sampling, or faseicular area and similar numbers of perfused microvessels were identified in each group for calculation of luminal areas. Total luminal area of endoneurial microvessels, mean microvessel area, mean microvessel diameter, mean microvessel perimeter and the diameter of the largest microvessel in each fascicle were all significantly greater in guanethidine animals than in controls (Table III and Fig. 2). To exclude a possible effect of the type of anaesthesia, each animal group included 6 pentobarbital-anaesthetized animals and 6 Inactin-anaesthetized animals. Morphometric comparisons were also made between control and drug-treated animals in each of the anaesthesia groups. Sampled fascicular area and total capillary area were independent of anaesthesia type. In both the control and guanethidine-treated animals mean microvessel area was consistently larger in pentobarbital-anaesthetized animals than in Inactin-anaesthetized animals but a larger number of microvessels were perfused in the control Inactin group. Mean microvessel perimeter was larger in the control pentobarbital group than the Inactin group. Within each anaesthesia type, microvessels in guanethidine-treated animals had a larger mean perimeter, mean luminal area and total luminal area than of controls (Table IV). Norepinephrine measurements Norepinephrine content was considerably reduced in the guanethidine-treated group (Table II).
116 DISCUSSION These studies indicate that chronic sympathectomy results in elevated nerve blood flow (NBF), a fall in microvascular calculated resistance (MR) and dilated endoneurial microvessels. In the guanethidine-treated animals, the slow component of hydrogen clearance, which likely represents endoneurial flow, was elevated above values observed in the control sham-injected animals. Similarly flow values were higher than those observed in the sciatic nerve of normal rats in studies using hydrogen clearance or the distribution of iodoantipyrine 13'~5'22. The drug-treated animals gained less weight than controls, but studies in our laboratory have not identified significant variations in HC with animal weights between 280 and 400 g. NBF following sympathectomy was similar to values noted following intra-arterial phentolamine 24. Guanethidine-treated animals had a fall in MAP, but a rise in NBF yielding a lower value of calculated MR. Phentolamine induced a similar effect 24. In the sciatic nerve of non-sympathectomized animals, NBF and MAP have a positive linear correlation - - a fall in MAP would be expected to reduce NBF. The findings of dilated India Ink-perfused microvesseis and loss of NE suggest that lower MR was a result of microvessel dilation deprived of NE-mediated vasoconstriction. We have previously identified that adrenergic NE-mediated vasoconstriction exists by observing shutoff of NBF following local intra-arterial NE delivery. Whole nerve NE is likely derived from adrenergic terminals on vasa nervorum. Similar loss of NE was observed in the vagus, tibial, sural, and peroneal nerves and the cervical sympathetic trunk of rats given guanethidine sulfate in a previous study 23. A non-specific toxic effect of guanethidine does not appear to account for the findings. In this study and previous investigations 23, somatic conduction was not influenced by guanethidine or by the accompanying reduction in weight gain. Guanethidine has a selective effect, sparing cholinergic and somatic nerves 9"14. Technical factors did not permit morphometric analysis of epineurial vessels. Epineurial vessels may be the critical sites of adrenergic influence, determining 'downstream' endoneurial perfusion 24. NE terminals have been noted to be confined to epineurial and perineurial vessels16. Our observations of dilated endoneurial microvessels may imply: (i) that these vessels are also directly innervated by the adrenergic system; (ii) that endoneurial microvessels dilate REFERENCES 1 Altura, A.T. and Altura, B.M., Barbiturates and aortic and venous smooth-muscle function, Anesthesiology, 43 (1975) 432-444. 2 Appenzeller, O., Dhital, K.K., Cowen, T. and Burnstock, G.,
under the conditions of increased 'feeding' flow from the epineurium; and (iii) that NE within the lumen of vessels travels 'downstream' from an epineurial origin into endoneurial vessels, Pericytes with contractile function surround some endoneurial capillaries. An endothelial receptor for NE has been postulated 3. Numbers of India Ink-perfused vessels did not differ between the groups likely excluding significant capillary recruitment in elevating NBF 12. Inactin anaesthesia, which is longer-acting and provides a more stable preparation, resulted in greater numbers of perfused microvessels with a smaller mean caliber than animals treated with pentobarbital. The similar fascicular size would tend to exclude a nonspecific effect on section preparation. A statistically significant difference in NBF between the guanethidine and control subgroups anaesthetized with pentobarbital was not observed despite a trend in the same direction as the Inactin group. A less stable level of anaesthesia with shorter-acting pentobarbital may have accounted for more variable NBF and MAP readings in the guanethidine group. MR, however, was lower in guanethidinetreated animals in both anaesthesia subgroups. Mean microvessels area, total capillary area and mean microvessel perimeter was greater in the guanethidine-treated group irrespective of anaesthesia effects. Circulating catecholamine levels may be higher in the pentobarbitalanaesthetized animals because of a more variable depth of anaesthesia. Circulating catecholamines, a direct drug effect 1 or other factors may have influenced microvessel recruitment and caliber to account for the changes observed, but the precise explanation is unclear. The regulatory characteristics of NBF are different from CBF, which is maintained by autoregulation H. Adrenergic innervation of cerebral vessels has been identified, but appears to have little influence on flow 1~. NBF, on the other hand, has negligible autoregulation 13"2°'21but appears to be influenced by the level of adrenergic 'tone', also a feature of other vascular beds4'6'7. There are likely other mechanisms of NBF regulation, but they are largely unexplored. Serotonin appears to obliterate AV shunt NBF TM and peptidergic influences on NBF have not been examined. Acknowledgements. The authors would like to acknowledge the invaluable technical assistance of James Schmelzerand Paula Zollman. Lisa Fregeau provided expert secretarial assistance. Ciba-Geigykindly provided guanethidine monosulfate. The research was supported in part by grants from NINCDS (R01 NS2 2352, NS14302), MDA, Mogg and Mayo Funds. P.A.L. is the recipient of a Jacob Javits Neuroscience Award. D.W.Z. receivedresearch fellowshipsupport from the Medical Research Council of Canada.
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