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In vitro study of acute elevations of endoneurial pressure in mammalian peripheral nerve sheath
EXPERIMENTAL
74, 160-169
NEUROLOGY
(1981)
ln vitro Study of Acute Elevations
in Mammalian
Peripheral PHILLIP
Neurophysiology Mayo Received
Laboratory, Foundation,
February
of Endoneurial Nerve Sheath
Pressure
A. Low’
Peripheral Nerve Center, Mayo Rochester, Minnesota 55905
5. 1981: revision
received
March
Clinic
and
23. I981
In previous studies of peripheral nerve edema, endoneurial fluid pressure rarely exceeded 7 mm Hg for even the most edematous nerves. In a recent study a timedependent reduction in elastic modulus was reported. However, because of the chronic nature of the studies, pressure and volume changes were not directly recorded from the same nerves. In the present study mammaliam perineurial compliance was directly studied in vitro. When compliance (AV/AP) was compared between nerves subjected to slow rates of AV (0.7 pl/min) and fast rates (7 aI/ min), mean values were reduced for all pressure intervals but did not become statistically significant because of considerable variability between nerves. However, in a separate series of experiments, when the same nerve sheath was consecutively subjected to these two rates of A V, compliance was consistently and significantly (P < 0.001, paired t test) greater for slow AV, confirming directly the importance of a time-dependent change in elastic modulus, i.e., a viscous modulus. A tentative physiologic-morphologic correlation is made. No evidence was found for norepinephrineor acetylcholine-responsive contractile elements.
INTRODUCTION The mammalian peripheral nerve is a fluid-containing tube encased in a perineurial sheath. Recently it was possible to measure endoneurial fluid pressure (EFP) using an active servonull system (7). The EFP was found to be increased in certain pathologic states such as experimental lead neuAbbreviation: EFP-endoneurial fluid pressure. ’ The investigator acknowledges with pleasure the helpful discussions with Drs. David Donald and Peter Dyck, Departments of Physiology and Biophysics, and Neurology, Mayo Clinic and Mayo Foundation, Rochester, Minn. This investigation was supported in part by a Peripheral Neuropathy Clinical Center Grant from the National Institute of Neurological and Communicative Disorders and Stroke (NS14304) and by Mayo Funds.
160 0014-4886/81/100160-10$02.00/O Copyright 0 198 I by Academic Press, Inc. All rights of reproduction in any form reserved
ACUTE
ELEVATIONS
OF ENDONEURIAL
PRESSURE
161
FIG. 1. Physiologic arrangement. A-inflow cannula. B-nerve sheath, C-outlet cannula, D-polyethylene tubing, E-AC Wheatstone bridge, F-micropipet, G-bellows system.
ropathy (5), Wallerian degeneration (9), and experimental galactose neuropathy (6). In these and other studies EFP did not exceed 7 mm Hg, pressures that would not collapse capillaries and hence would not be expected to produce severe ischemia to nerve. Relevant to the above observation is the finding of a time-dependent reduction in elastic modulus (6). However, in certain situations such as heat or cold injury to nerve (14) and ,bleeding into nerve (4), the nerve swells much more rapidly and the pressure-volume relationships at these early times are not known. In this study we examined acute volume increment in vitro and addressed the question of time-dependent changes in perineurial compliance directly. Perineurial components resembling smooth muscle have been described (10) and contractile properties suggested. We tested the responsiveness of these components to pharmacologic agents with vasoconstrictor and vasodilator properties. METHODS Preparation. My studies were conducted on male Sprague-Dawley rats (approximately 300 g). The animal was killed by a blow on the head and the sciatic nerve removed and stored in oxygenated mammalian Ringer’s solution until use (145 RIM NaCl, 3.5 mM KCl, 2.0 mM CaClz, glucose, 5mM Hepes buffer, pH 7.4, osmolarity 290 mosM). The nerve sheath was isolated atraumatically as follows. Only short segments were used (3 to 4 mm). The sheath was not handled directly. Instead the nerve was decompressed by extracting a few fibers using a fine forceps while a fascicle on the opposite side was held by another forceps. The process was continued until >90% of the fibers were removed. The Physiolog-ical Arrangement (Fig. I). The chamber consisted of a 14 x 40 x 6-mm Lucite chamber with inflow and outflow ducts. The inlet
162
PHILLIP
A. LOW
cannula consisted of a stainless-steel cannula (A, external diameter 640 pm)connected to a tuberculin syringe. The outlet cannula consisted of a short, wider-bore cannula (C; external diameter 900 pm). The distance between the cannulas was 1.4 mm. The infusate was rat plasma containing 0.2% Evans blue and was driven by an infusion pump with variable rate control (Harvard infusion pump Model 902). The nerve sheath (B) was fitted over the two cannulas and tied on each end with two 6-O black silk sutures. The EFP was measured using a glass micropipet (F) (0.1 to 0.2 MB) containing 0.5 M saline. The micropipet was wedged into a short length of polyethylene tubing (D) connected to the outlet cannulas. Pressure recordings were measured using a modification of an active servonull device originally developed by Wiederhielm ( 16). In brief, the micropipet contains hyperosmolar saline and forms one arm of a balanced AC wheatstone bridge (E). When the micropipet is wedged into the tubing, the EFP causes a shift in the fluid interface generating an error voltage which is amplified and applied to a bellows system (G) which generates a counterpressure within the pipet returning the interface to its original position. The hydraulic pressure was measured with a transducer and recorded on a chart recorder. The preparation was superfused with oxygenated mammalian Ringer’s solution (PH 7.4, osmolarity 290 mosM) maintained at 37°C by a temperature-controlled bath (Sybron-Thermolyne). The solution was pumped into the chamber (Model 203 peristaltic pump, Scientific Industries, Inc.). Excess solution was aspirated via the outflow duct which also controlled the fluid level. Data Analysis. Volume increments for the following pressure increments were measured from the chart recorder: O-l, l-3, 3-5, 5-7, 7- 10, 10-l 5, 15-50 mm Hg, and the data entered into a computer (Hewlett-Packard 9845B) for further data reduction. Because the same AV in nerves of different diameters would cause different AP, the nerves were all normalized to a radius of 500 pm as follows: V = rR=L,
and since z and L are constant, V a R2. Example: For a nerve of 450 pm, VN = V, X (500/450)=, where R = radius, L = length, V, = original volume, and V, = normalized volume. The data were subsequently reduced in two ways: (i) curves were fitted using the Marquardt algorithm for nonlinear regression (HP nonlinear regression package); (ii) compliances of a-ylAP for specified pressure in-
ACUTE
ELEVATIONS
VOLUME
OF ENDONEURIAL
INCREMENT
PRESSURE
163
(~1)
FIG. 2. Pressure-volume curve. Infusion rate 7 pl/min, into nerve sheath radius 450 pm. Temperature 37°C. Curve fitted using third-degree polynomial computer generated and plotted. A, B, C are segments of curve detailed in text.
tervals were separately determined. Student’s c test for paired and grouped data was used. Sources of Error and Their Minimization. (i) Error due to longitudinal stretch: With volume increment there is longitudinal as well as circumferential stretch. The longitudinal stretch was minimized and standardized by maintaining a short and constant length of perineurium (2.4 mm). (ii) Error due to fluid leak: Potential sites of leakage are the sheath, the ties, and the exit cannula. Nerve sheath leakage was monitored under a dissecting microscope for leakage of Evans blue-albumin. Leakage did not occur under the ties. At high pressures when leakage occurred it did so through the perineurium rather than under the tie. The micropipet-polyethylene tubing seal was leakproof. With the servonull mechanism there is no net movement of fluid into the micropipet. (iii) Error due to pressure gradient: With rapid rates of infusion Pascal’s law (pressure is uniform everywhere within a fluid-filled tube) may not apply because a pressure gradient may occur. To minimize this source of error the outlet cannula was kept short (25 mm) and its diameter (900 pm). larger than that of the inlet cannula (640 pm). (iv) Error due to nerves of different diameters: Nerve diameter was
164
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measured using a micrometer eyepiece (X32). Because the nerve sheath collapses with removal of nerve fibers, the diameter was determined at a pressure of 1 mm Hg (its physiologic pressure) (5, 7). The AV was then normalized for a nerve of diameter 500 pm to obviate errors due to different nerve diameters. RESULTS Thirty-one nerves were studied with mean radius 448 pm, SD 65 pm. Serum was infused at 0.7 bl/min (slow rate) or 7 pl/min (fast rate). To obviate problems inherent in infusing solution in nerves of different diameters all nerves were normalized to a radius of 500 pm (see Methods). With progressive volume increments (Fig. 2) there was initially a phase of volume increase with very small pressure rise (A) followed by a curvilinear and increasing AP/AV relationship (B), in turn followed by a linear AP/AV segment. Leakage of Evans blue-labeled serum was not seen in segments A and B. It was seen in some nerves when pressure exceeded 40 mm Hg. Perineurial distensibility was reversible for pressure increments ~20 mm Hg but less reversible for larger pressure changes. Diameter changes were largest in A, moderate in B, and small in C. In the qualitative part of the study, the volume was held constant in five
MEFIN FIG. 3. Pressure-volume fourth-degree polynomial; mial, computer generated
VOLUME
INCREMENT
curve for grouped data. Left-(infusion right-(infusion rate 0.7 rl/min) fitted and plotted.
(~11 rate 7 pl/min) fitted with with third-degree polyno-
ACUTE
ELEVATIONS
OF
ENDONEURIAL
165
PRESSURE
nerves at small volumes (the AV required to generate a AP of 5 mm Hg) and large volumes (AV to generate 20 mm Hg). There was a negative AZ’ with time that was more prominent for initial pressures of 5 mm Hg than 20 mm Hg. For slow rates of volume increment the nerve sheath was more compliant. There was a lower AP/AV ratio for all intervals (Fig. 3). When compliance was expressed for different pressure intervals, mean compliance was greater for the slow rate of infusion (Fig. 4). However the differences were not significantly different for any of the pressure intervals (t > 0.05, Student’s 2 test). Because there appeared to be considerable variability in compliance in different nerves, a further series of experiments was done in which the effect of fast and slow rates of infusion was tested on the same sheaths. For this part of the experiment AP was not allowed to exceed 20 mm Hg and an interval of at least 10 min was provided between trials to ensure reversibility. Paired studies were made on eight nerves. In four nerves, the initial infusion rate was fast and in the remainder, slow. In all nerves compliance was consistently greater for the slow rate of infusion (Fig. 5) 7ul/min r-77rj
0.7ul/min m
COMWANCE(nl/mmHg)
250
200
150
100
50
0 1-1OmmHg
lo-2OmmHg
PRESSURE FIG. 4. Compliance significant (t > 0.05,
JO-4OmmHp
20-3OmmHg
INTERVALS
(mm/Hg)
for pressure intervals indicated. Bars Student’s t test for grouped data).
represent
1
SEM.
Difference
not
166
PHILLlP
A. LOW
I
r Z w
24
5 5 -
15
*
1 *t l *
#1
8
#2
#3
#4
PERINEURIAL
#5
#6
17
SHEATHS
#a
(by #)
FIG. 5. Compliance for paired study. The increase in compliance is expressed as percentage increase over the fast rate of infusion. significant (t < 0.001, paired t test).
for the slow rate of infusion The differences are highly
and for all pressure intervals ‘the difference was highly significant (P < 0.001, paired t test). The effect of acetylcholine (20 FM four nerves; 200 pM, two nerves) in mammalian Ringer’s solution was superfused on nerve sheath distended to 2 mm Hg. No change in volume or pressure was observed during the test period of 60 min. Norepinephrine (40 PM, four nerves; 400 PM, two nerves) in mammalian =z======VOLUME taking unstressed
up
INCREMENT========>>
of volume
of
m-orientation elastin k
collagen taking perineurial
up
stretch
of overlap of
elastin stretch
of
perimurial
stretch
<====A====>
FIG. 6. Morphologic-physiologic C of pressure:volume curve (Fig
cell
of collagen
<====B====><===C====>
correlations 2).
based on published
data
for segments
A, B,
ACUTE
ELEVATIONS
OF ENDONEURIAL
PRESSURE
167
Ringer’s solution also did not change the nerve sheath pressure’or volume during the test lasting 1 h. DISCUSSION In previous reports of EFP, the magnitude rarely exceeded 7 mm Hg (5, 6, 9) in which EFP was recorded hours, days, or months after commencement of the studies. The importance of time-dependent AP/AV changes was suggested in a previous study (6). In that report volume and pressure were not simultaneously recorded. The present study directly confirms the importance of a time-dependent change in compliance, i.e., in nerve sheath there is an elastic modulus and a viscous modulus (rate of change of elastic modulus) (8). Compared with the AP/AV of blood vessels, the curves are qualitatively similar but shifted toward a lower AP/AV part of the curve. In blood vessels AP/AV is linear for pressures > 70 to 100 mm Hg ( 1) whereas for nerve AP/AV is linear at pressures > 10 to 20 mm Hg. The findings of a time-dependent increase in compliance may be related to stress relaxation [less stress (AP) is required with time to produce the same deformation (Av] and creep (12) [with time the same stress (AP) produces progressively greater deformation (Av)]. During the qualitative part of the study, when volume was held constant there was indeed a decrease in pressure with time and the time-dependent decrease in EFP was more prominent at small volumes than large, indicating greater stress relaxation at low volumes (segment B, Fig. 2) than high volumes (segment C, Fig. 2). The diameter measurements suggest a component of creep. However, the present study with AV as the independent variable was not designed to rigorously quantitate creep behavior. The present findings of a larger modulus in acute studies than in chronic experiments have important applications. Very acute swelling of mammalian nerve trunk may occur in intraneurial hemorrhage (14) and peripheral nerve injury due to heat and cold (6). Under these circumstances the sustained pressure elevation of >20 mm Hg would be expected to collapse capillaries causing ischemic necrosis of nerve fibers. The nerve sheath used in the present study consisted of perineurium and some epineurium. Epineurium contains collagen bundles largely orientated longitudinally (13). There is also some elastin whose orientation is also mostly longitudinal. The perineurium consists of polygonal cells which overlap and interdigitate with one another (2, 13) and are encased in basal lamina. Associated with these polygonal cells are collagen and elastin fibrils orientated in a loose lattice-like fashion (15). Morphological studies were not carried out (in the present study) so that
168
PHILLIP
A. LOW
a direct physiologic-morphologic correlate is not possible. However, correlating the present findings with published data, the following is a tentative interpretation of the data and provides a basis for future testing (Fig 6). For small volume increments (A) AP/AV is small and ascribed to the taking up of unstressed volume (11). An additional component may be the reorientation of loosely orientated fibrils. For further volume increments there is an incremental AP/AV (B). This part of the AP/AV curve is reversible, and perineurial leakage does not occur and is associated with progressive increase in diameter. For these reasons the morphologic basis of the observed changes has been ascribed to the reduction in perineurial cellular overlap and elasticity of circumferentially orientated elastin fibers. In part C of the curve there is only a very small increase in diameter, the AP/AV relationship is approximately linear, and perineurial leakage may occur. This “stiff’ portion of the curve is analogous to the linear part of the AP/AV curve in blood vessels whose morphologic basis is known to be that of collagen and not elastin (3). Because perineurial circumferentially orientated collagen is relatively scant compared with blood vessels, a role for perineurial cellular stretch is also implicated. A role for the perineurial cell as a contractile cell has been postulated (4). The investigators noted the resemblance to peritubular contractile cells of mice testis and described bundles of closely aggregated filaments (with “attachment devices”) similar in appearance to those of myofilaments of smooth muscle. We were unable to demonstrate a contractile response pharmacologically. Contraction or relaxation causing either longitudinal or circumferential dimensional changes would have been detected as a pressure change. The diameter was also monitored and not noted to change. REFERENCES I. BADER, H. 1967. Dependence of wall stress in the human thoracic aorta on age and pressure. Circ. Res. 20: 354-361. 2. CRAVIOTO, H. 1966. The perineurium as a diffusion barrier-ultrastructural correlates. Bull.
Los Angeles
Neurol.
Sot. 31: 106-208.
3. DOBRIN, P. B. 1978. Mechanical properties of arteries. Physiol. Rev. 58: 397-456. 4. DYCK, P. J. 1975. Pathologic alterations of the peripheral nervous system of man. Pages 296-336 in P. J. DYCK, P. K. THOMAS, AND E. H. LAMBERT, Eds., Peripheral Neuropathy, Saunders, Philadelphia. 5. LOW, P. A., AND P. J. DYCK. 1977. Increased endoneurial fluid pressure in experimental lead neuropathy. Nature (London) 269: 427-428. 6. Low, P. A., P. J. DYCK, AND J. D. SCHMELZER. 1980. Mammalian peripheral nerve sheath has unique responses to chronic elevations of endoneurial fluid pressure. Exp. Neural. 7.
70: 300-306.
Low, P. A., G. MARCHAND, F. KNOX, AND P. J. DYCK. 1977. Measurement of endoneurial fluid pressure with polyethylene matrix capsules. Brain Res. 122: 373-377.
ACUTE
ELEVATIONS
OF ENDONEURIAL
PRESSURE
169
8. PETERSON, L. H., R. E. JENSEN, AND J. PARNELL. 1960. Mechanical properties of arteries in vivo. Circ. Res. 8: 622-639. 9. POWELL, H. C., R. R. MEYERS, M. L. COSTELLO, AND P. W. LAMPERT. 1979. Endoneurial fluid pressure in Wallerian degeneration. Ann. Neurof. 5: 550-557. 10. ROSS, M. H., AND E. J. REITH. 1969. Perineurium: evidence for contractile elements. Science 165: 604-606. 11. ROTHE, C. F. 1978. Reflex control of the veins in cardiovascular function. Tutorial lecture presented at the American Physiological Society Fall Meeting, Oct. 23, 1978, St. Louis, MO. 12. SARNOFF, S. J., AND E. BERGLUND. 1952. Pressure-volume characteristics and stress relaxation in the pulmonary vascular bed of the dog. Am. J. Physiol. 171: 238. 13. THOMAS, P. K. 1963. The connective tissue of peripheral nerve: an electron microscope study. J. Amt. 97: 35-44. 14. THOMAS, P. K., AND J. B. CAVANAGH. 1975. Neuropathy due to physical agents. Pages 734-754 in P. J. DYCK. P. K. THOMAS, AND E. H. LAMBERT, Eds., Peripheral Neuropathy, Saunders, Philadelphia. 15. THOMAS, P. K., AND Y. OUSON. 1975. Microscopic anatomy and function of the connective tissue components of peripheral nerve. Pages 168-189 in P. J. DYCK, P. K. THOMAS, AND E. H. LAMBERT, Eds., Peripheral Neuropathy, Saunders, Philadelphia. 16. WIEDERHIELM, C. A., J. W. WOODBURY, S. KIRK, AND R. F. RUSHMER. 1964. Pulsatile pressures in the microcirculation of frog’s mesentery. Am. J. Physiol. 20’1: 173-176.
74, 160-169
NEUROLOGY
(1981)
ln vitro Study of Acute Elevations
in Mammalian
Peripheral PHILLIP
Neurophysiology Mayo Received
Laboratory, Foundation,
February
of Endoneurial Nerve Sheath
Pressure
A. Low’
Peripheral Nerve Center, Mayo Rochester, Minnesota 55905
5. 1981: revision
received
March
Clinic
and
23. I981
In previous studies of peripheral nerve edema, endoneurial fluid pressure rarely exceeded 7 mm Hg for even the most edematous nerves. In a recent study a timedependent reduction in elastic modulus was reported. However, because of the chronic nature of the studies, pressure and volume changes were not directly recorded from the same nerves. In the present study mammaliam perineurial compliance was directly studied in vitro. When compliance (AV/AP) was compared between nerves subjected to slow rates of AV (0.7 pl/min) and fast rates (7 aI/ min), mean values were reduced for all pressure intervals but did not become statistically significant because of considerable variability between nerves. However, in a separate series of experiments, when the same nerve sheath was consecutively subjected to these two rates of A V, compliance was consistently and significantly (P < 0.001, paired t test) greater for slow AV, confirming directly the importance of a time-dependent change in elastic modulus, i.e., a viscous modulus. A tentative physiologic-morphologic correlation is made. No evidence was found for norepinephrineor acetylcholine-responsive contractile elements.
INTRODUCTION The mammalian peripheral nerve is a fluid-containing tube encased in a perineurial sheath. Recently it was possible to measure endoneurial fluid pressure (EFP) using an active servonull system (7). The EFP was found to be increased in certain pathologic states such as experimental lead neuAbbreviation: EFP-endoneurial fluid pressure. ’ The investigator acknowledges with pleasure the helpful discussions with Drs. David Donald and Peter Dyck, Departments of Physiology and Biophysics, and Neurology, Mayo Clinic and Mayo Foundation, Rochester, Minn. This investigation was supported in part by a Peripheral Neuropathy Clinical Center Grant from the National Institute of Neurological and Communicative Disorders and Stroke (NS14304) and by Mayo Funds.
160 0014-4886/81/100160-10$02.00/O Copyright 0 198 I by Academic Press, Inc. All rights of reproduction in any form reserved
ACUTE
ELEVATIONS
OF ENDONEURIAL
PRESSURE
161
FIG. 1. Physiologic arrangement. A-inflow cannula. B-nerve sheath, C-outlet cannula, D-polyethylene tubing, E-AC Wheatstone bridge, F-micropipet, G-bellows system.
ropathy (5), Wallerian degeneration (9), and experimental galactose neuropathy (6). In these and other studies EFP did not exceed 7 mm Hg, pressures that would not collapse capillaries and hence would not be expected to produce severe ischemia to nerve. Relevant to the above observation is the finding of a time-dependent reduction in elastic modulus (6). However, in certain situations such as heat or cold injury to nerve (14) and ,bleeding into nerve (4), the nerve swells much more rapidly and the pressure-volume relationships at these early times are not known. In this study we examined acute volume increment in vitro and addressed the question of time-dependent changes in perineurial compliance directly. Perineurial components resembling smooth muscle have been described (10) and contractile properties suggested. We tested the responsiveness of these components to pharmacologic agents with vasoconstrictor and vasodilator properties. METHODS Preparation. My studies were conducted on male Sprague-Dawley rats (approximately 300 g). The animal was killed by a blow on the head and the sciatic nerve removed and stored in oxygenated mammalian Ringer’s solution until use (145 RIM NaCl, 3.5 mM KCl, 2.0 mM CaClz, glucose, 5mM Hepes buffer, pH 7.4, osmolarity 290 mosM). The nerve sheath was isolated atraumatically as follows. Only short segments were used (3 to 4 mm). The sheath was not handled directly. Instead the nerve was decompressed by extracting a few fibers using a fine forceps while a fascicle on the opposite side was held by another forceps. The process was continued until >90% of the fibers were removed. The Physiolog-ical Arrangement (Fig. I). The chamber consisted of a 14 x 40 x 6-mm Lucite chamber with inflow and outflow ducts. The inlet
162
PHILLIP
A. LOW
cannula consisted of a stainless-steel cannula (A, external diameter 640 pm)connected to a tuberculin syringe. The outlet cannula consisted of a short, wider-bore cannula (C; external diameter 900 pm). The distance between the cannulas was 1.4 mm. The infusate was rat plasma containing 0.2% Evans blue and was driven by an infusion pump with variable rate control (Harvard infusion pump Model 902). The nerve sheath (B) was fitted over the two cannulas and tied on each end with two 6-O black silk sutures. The EFP was measured using a glass micropipet (F) (0.1 to 0.2 MB) containing 0.5 M saline. The micropipet was wedged into a short length of polyethylene tubing (D) connected to the outlet cannulas. Pressure recordings were measured using a modification of an active servonull device originally developed by Wiederhielm ( 16). In brief, the micropipet contains hyperosmolar saline and forms one arm of a balanced AC wheatstone bridge (E). When the micropipet is wedged into the tubing, the EFP causes a shift in the fluid interface generating an error voltage which is amplified and applied to a bellows system (G) which generates a counterpressure within the pipet returning the interface to its original position. The hydraulic pressure was measured with a transducer and recorded on a chart recorder. The preparation was superfused with oxygenated mammalian Ringer’s solution (PH 7.4, osmolarity 290 mosM) maintained at 37°C by a temperature-controlled bath (Sybron-Thermolyne). The solution was pumped into the chamber (Model 203 peristaltic pump, Scientific Industries, Inc.). Excess solution was aspirated via the outflow duct which also controlled the fluid level. Data Analysis. Volume increments for the following pressure increments were measured from the chart recorder: O-l, l-3, 3-5, 5-7, 7- 10, 10-l 5, 15-50 mm Hg, and the data entered into a computer (Hewlett-Packard 9845B) for further data reduction. Because the same AV in nerves of different diameters would cause different AP, the nerves were all normalized to a radius of 500 pm as follows: V = rR=L,
and since z and L are constant, V a R2. Example: For a nerve of 450 pm, VN = V, X (500/450)=, where R = radius, L = length, V, = original volume, and V, = normalized volume. The data were subsequently reduced in two ways: (i) curves were fitted using the Marquardt algorithm for nonlinear regression (HP nonlinear regression package); (ii) compliances of a-ylAP for specified pressure in-
ACUTE
ELEVATIONS
VOLUME
OF ENDONEURIAL
INCREMENT
PRESSURE
163
(~1)
FIG. 2. Pressure-volume curve. Infusion rate 7 pl/min, into nerve sheath radius 450 pm. Temperature 37°C. Curve fitted using third-degree polynomial computer generated and plotted. A, B, C are segments of curve detailed in text.
tervals were separately determined. Student’s c test for paired and grouped data was used. Sources of Error and Their Minimization. (i) Error due to longitudinal stretch: With volume increment there is longitudinal as well as circumferential stretch. The longitudinal stretch was minimized and standardized by maintaining a short and constant length of perineurium (2.4 mm). (ii) Error due to fluid leak: Potential sites of leakage are the sheath, the ties, and the exit cannula. Nerve sheath leakage was monitored under a dissecting microscope for leakage of Evans blue-albumin. Leakage did not occur under the ties. At high pressures when leakage occurred it did so through the perineurium rather than under the tie. The micropipet-polyethylene tubing seal was leakproof. With the servonull mechanism there is no net movement of fluid into the micropipet. (iii) Error due to pressure gradient: With rapid rates of infusion Pascal’s law (pressure is uniform everywhere within a fluid-filled tube) may not apply because a pressure gradient may occur. To minimize this source of error the outlet cannula was kept short (25 mm) and its diameter (900 pm). larger than that of the inlet cannula (640 pm). (iv) Error due to nerves of different diameters: Nerve diameter was
164
PHILLIP
A. LOW
measured using a micrometer eyepiece (X32). Because the nerve sheath collapses with removal of nerve fibers, the diameter was determined at a pressure of 1 mm Hg (its physiologic pressure) (5, 7). The AV was then normalized for a nerve of diameter 500 pm to obviate errors due to different nerve diameters. RESULTS Thirty-one nerves were studied with mean radius 448 pm, SD 65 pm. Serum was infused at 0.7 bl/min (slow rate) or 7 pl/min (fast rate). To obviate problems inherent in infusing solution in nerves of different diameters all nerves were normalized to a radius of 500 pm (see Methods). With progressive volume increments (Fig. 2) there was initially a phase of volume increase with very small pressure rise (A) followed by a curvilinear and increasing AP/AV relationship (B), in turn followed by a linear AP/AV segment. Leakage of Evans blue-labeled serum was not seen in segments A and B. It was seen in some nerves when pressure exceeded 40 mm Hg. Perineurial distensibility was reversible for pressure increments ~20 mm Hg but less reversible for larger pressure changes. Diameter changes were largest in A, moderate in B, and small in C. In the qualitative part of the study, the volume was held constant in five
MEFIN FIG. 3. Pressure-volume fourth-degree polynomial; mial, computer generated
VOLUME
INCREMENT
curve for grouped data. Left-(infusion right-(infusion rate 0.7 rl/min) fitted and plotted.
(~11 rate 7 pl/min) fitted with with third-degree polyno-
ACUTE
ELEVATIONS
OF
ENDONEURIAL
165
PRESSURE
nerves at small volumes (the AV required to generate a AP of 5 mm Hg) and large volumes (AV to generate 20 mm Hg). There was a negative AZ’ with time that was more prominent for initial pressures of 5 mm Hg than 20 mm Hg. For slow rates of volume increment the nerve sheath was more compliant. There was a lower AP/AV ratio for all intervals (Fig. 3). When compliance was expressed for different pressure intervals, mean compliance was greater for the slow rate of infusion (Fig. 4). However the differences were not significantly different for any of the pressure intervals (t > 0.05, Student’s 2 test). Because there appeared to be considerable variability in compliance in different nerves, a further series of experiments was done in which the effect of fast and slow rates of infusion was tested on the same sheaths. For this part of the experiment AP was not allowed to exceed 20 mm Hg and an interval of at least 10 min was provided between trials to ensure reversibility. Paired studies were made on eight nerves. In four nerves, the initial infusion rate was fast and in the remainder, slow. In all nerves compliance was consistently greater for the slow rate of infusion (Fig. 5) 7ul/min r-77rj
0.7ul/min m
COMWANCE(nl/mmHg)
250
200
150
100
50
0 1-1OmmHg
lo-2OmmHg
PRESSURE FIG. 4. Compliance significant (t > 0.05,
JO-4OmmHp
20-3OmmHg
INTERVALS
(mm/Hg)
for pressure intervals indicated. Bars Student’s t test for grouped data).
represent
1
SEM.
Difference
not
166
PHILLlP
A. LOW
I
r Z w
24
5 5 -
15
*
1 *t l *
#1
8
#2
#3
#4
PERINEURIAL
#5
#6
17
SHEATHS
#a
(by #)
FIG. 5. Compliance for paired study. The increase in compliance is expressed as percentage increase over the fast rate of infusion. significant (t < 0.001, paired t test).
for the slow rate of infusion The differences are highly
and for all pressure intervals ‘the difference was highly significant (P < 0.001, paired t test). The effect of acetylcholine (20 FM four nerves; 200 pM, two nerves) in mammalian Ringer’s solution was superfused on nerve sheath distended to 2 mm Hg. No change in volume or pressure was observed during the test period of 60 min. Norepinephrine (40 PM, four nerves; 400 PM, two nerves) in mammalian =z======VOLUME taking unstressed
up
INCREMENT========>>
of volume
of
m-orientation elastin k
collagen taking perineurial
up
stretch
of overlap of
elastin stretch
of
perimurial
stretch
<====A====>
FIG. 6. Morphologic-physiologic C of pressure:volume curve (Fig
cell
of collagen
<====B====><===C====>
correlations 2).
based on published
data
for segments
A, B,
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Ringer’s solution also did not change the nerve sheath pressure’or volume during the test lasting 1 h. DISCUSSION In previous reports of EFP, the magnitude rarely exceeded 7 mm Hg (5, 6, 9) in which EFP was recorded hours, days, or months after commencement of the studies. The importance of time-dependent AP/AV changes was suggested in a previous study (6). In that report volume and pressure were not simultaneously recorded. The present study directly confirms the importance of a time-dependent change in compliance, i.e., in nerve sheath there is an elastic modulus and a viscous modulus (rate of change of elastic modulus) (8). Compared with the AP/AV of blood vessels, the curves are qualitatively similar but shifted toward a lower AP/AV part of the curve. In blood vessels AP/AV is linear for pressures > 70 to 100 mm Hg ( 1) whereas for nerve AP/AV is linear at pressures > 10 to 20 mm Hg. The findings of a time-dependent increase in compliance may be related to stress relaxation [less stress (AP) is required with time to produce the same deformation (Av] and creep (12) [with time the same stress (AP) produces progressively greater deformation (Av)]. During the qualitative part of the study, when volume was held constant there was indeed a decrease in pressure with time and the time-dependent decrease in EFP was more prominent at small volumes than large, indicating greater stress relaxation at low volumes (segment B, Fig. 2) than high volumes (segment C, Fig. 2). The diameter measurements suggest a component of creep. However, the present study with AV as the independent variable was not designed to rigorously quantitate creep behavior. The present findings of a larger modulus in acute studies than in chronic experiments have important applications. Very acute swelling of mammalian nerve trunk may occur in intraneurial hemorrhage (14) and peripheral nerve injury due to heat and cold (6). Under these circumstances the sustained pressure elevation of >20 mm Hg would be expected to collapse capillaries causing ischemic necrosis of nerve fibers. The nerve sheath used in the present study consisted of perineurium and some epineurium. Epineurium contains collagen bundles largely orientated longitudinally (13). There is also some elastin whose orientation is also mostly longitudinal. The perineurium consists of polygonal cells which overlap and interdigitate with one another (2, 13) and are encased in basal lamina. Associated with these polygonal cells are collagen and elastin fibrils orientated in a loose lattice-like fashion (15). Morphological studies were not carried out (in the present study) so that
168
PHILLIP
A. LOW
a direct physiologic-morphologic correlate is not possible. However, correlating the present findings with published data, the following is a tentative interpretation of the data and provides a basis for future testing (Fig 6). For small volume increments (A) AP/AV is small and ascribed to the taking up of unstressed volume (11). An additional component may be the reorientation of loosely orientated fibrils. For further volume increments there is an incremental AP/AV (B). This part of the AP/AV curve is reversible, and perineurial leakage does not occur and is associated with progressive increase in diameter. For these reasons the morphologic basis of the observed changes has been ascribed to the reduction in perineurial cellular overlap and elasticity of circumferentially orientated elastin fibers. In part C of the curve there is only a very small increase in diameter, the AP/AV relationship is approximately linear, and perineurial leakage may occur. This “stiff’ portion of the curve is analogous to the linear part of the AP/AV curve in blood vessels whose morphologic basis is known to be that of collagen and not elastin (3). Because perineurial circumferentially orientated collagen is relatively scant compared with blood vessels, a role for perineurial cellular stretch is also implicated. A role for the perineurial cell as a contractile cell has been postulated (4). The investigators noted the resemblance to peritubular contractile cells of mice testis and described bundles of closely aggregated filaments (with “attachment devices”) similar in appearance to those of myofilaments of smooth muscle. We were unable to demonstrate a contractile response pharmacologically. Contraction or relaxation causing either longitudinal or circumferential dimensional changes would have been detected as a pressure change. The diameter was also monitored and not noted to change. REFERENCES I. BADER, H. 1967. Dependence of wall stress in the human thoracic aorta on age and pressure. Circ. Res. 20: 354-361. 2. CRAVIOTO, H. 1966. The perineurium as a diffusion barrier-ultrastructural correlates. Bull.
Los Angeles
Neurol.
Sot. 31: 106-208.
3. DOBRIN, P. B. 1978. Mechanical properties of arteries. Physiol. Rev. 58: 397-456. 4. DYCK, P. J. 1975. Pathologic alterations of the peripheral nervous system of man. Pages 296-336 in P. J. DYCK, P. K. THOMAS, AND E. H. LAMBERT, Eds., Peripheral Neuropathy, Saunders, Philadelphia. 5. LOW, P. A., AND P. J. DYCK. 1977. Increased endoneurial fluid pressure in experimental lead neuropathy. Nature (London) 269: 427-428. 6. Low, P. A., P. J. DYCK, AND J. D. SCHMELZER. 1980. Mammalian peripheral nerve sheath has unique responses to chronic elevations of endoneurial fluid pressure. Exp. Neural. 7.
70: 300-306.
Low, P. A., G. MARCHAND, F. KNOX, AND P. J. DYCK. 1977. Measurement of endoneurial fluid pressure with polyethylene matrix capsules. Brain Res. 122: 373-377.
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8. PETERSON, L. H., R. E. JENSEN, AND J. PARNELL. 1960. Mechanical properties of arteries in vivo. Circ. Res. 8: 622-639. 9. POWELL, H. C., R. R. MEYERS, M. L. COSTELLO, AND P. W. LAMPERT. 1979. Endoneurial fluid pressure in Wallerian degeneration. Ann. Neurof. 5: 550-557. 10. ROSS, M. H., AND E. J. REITH. 1969. Perineurium: evidence for contractile elements. Science 165: 604-606. 11. ROTHE, C. F. 1978. Reflex control of the veins in cardiovascular function. Tutorial lecture presented at the American Physiological Society Fall Meeting, Oct. 23, 1978, St. Louis, MO. 12. SARNOFF, S. J., AND E. BERGLUND. 1952. Pressure-volume characteristics and stress relaxation in the pulmonary vascular bed of the dog. Am. J. Physiol. 171: 238. 13. THOMAS, P. K. 1963. The connective tissue of peripheral nerve: an electron microscope study. J. Amt. 97: 35-44. 14. THOMAS, P. K., AND J. B. CAVANAGH. 1975. Neuropathy due to physical agents. Pages 734-754 in P. J. DYCK. P. K. THOMAS, AND E. H. LAMBERT, Eds., Peripheral Neuropathy, Saunders, Philadelphia. 15. THOMAS, P. K., AND Y. OUSON. 1975. Microscopic anatomy and function of the connective tissue components of peripheral nerve. Pages 168-189 in P. J. DYCK, P. K. THOMAS, AND E. H. LAMBERT, Eds., Peripheral Neuropathy, Saunders, Philadelphia. 16. WIEDERHIELM, C. A., J. W. WOODBURY, S. KIRK, AND R. F. RUSHMER. 1964. Pulsatile pressures in the microcirculation of frog’s mesentery. Am. J. Physiol. 20’1: 173-176.