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Microvascular Blood Flow is Altered After Repair of Testicular Torsion in the Rat
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THE JOURNAL
Vol. 167. 1493-1498, April 1997 Printed in U S A .
OF UROLOCY
Copyright 0 1997 by h m c m U R O ~ I CASSOCIATION, AL hc.
MICROVASCULAR BLOOD FLOW IS ALTERED AFTER REPAIR OF TESTICULAR TORSION IN THE RAT E. J. BECKER, H. M. PRILLAMAN AND T. T. TURNER* From the Department of Urology and Department of Cell Biology, University of Virginia School of Medicine, Charlottesville, Virginia
ABSTRACT
Purpose: Previous studies have shown that experimental testicular torsion with a duration of 1hr. or longer causes irreversible damage to the rat testis, but that testicular blood flow values are normal 24 hrs. after repair of torsion. More acute evaluation of return blood flow after repair of torsion has not been performed and was the topic of this study. Materials and Methods: Laser-Doppler flowmetry was used to evaluate teaticular microvascular blood flow before application of 1,2, or 4 hr., 720" torsion, during torsion, and at several time points after repair of torsion. Experiments were performed in both adult and prepubertal rats. Results: Testicular torsion essentially eliminated blood flow in both adult and prepubertal testes. Considering all the flow data within each group after torsion repair, increasing time of torsion was associated with significantly less return blood flow in both adult and prepubertal animals. Interestingly, only the four hour torsion data was associated with reduced return flow in prepubertal animals while both two and four hour torsion were associated with poor return flow in adult animals. Vasomotion, or pulsatile microvascular flow, often seen before torsion in both adult and prepubertal animals, was never seen after torsion repair. Conclusions: Increasing times of torsion are associated with lower microvascular blood flow values during the hour following the relief of torsion. Vasomotion is eliminated by torsion during the period studied. Whether vasomotion returns is unknown, but altering this flow pattern might be involved in the mechanism of injury caused by acute torsion. KEY WORDS:micmvasculature, blood flow,testis, rat, vasomotion Testicular torsion is defined as a twisting of the spermatic fore, during, and immediately (5-60 minutes) after various cord which results in impedance of blood flow to the involved durations of torsion. Testicular torsion primarily affecta pen-pubertal males alorgan.l.2 The degree and duration of torsion in the clinical setting is widely variable. Work with various animal models though most of the research published concerning torsion has has demonstrated a clear relationship between duration of involved adult animal models. The prepubertal testis is still in terms of size, vascular architecture, and the torsion and the resultant damage to the torsed t e s t i ~ . ~developing .~ Conventional operative management calls for detorsion of complex cellular relationships which allow for the completion the testis and observation; testes which reperfuse are left in of spermatogenesis. Since the vascular consequences of tesplace and are pexed to the scrotal septum. Testes which ticular torsion might be different in prepubertal and adult appear infarcted are removed. The exact incidence of later testes, we have performed the present studies in both types of atrophy in "salvaged" testes is unknown but by most reports animals. appears to be quite high.5,6,7 Clinical data concerning the relative function of these atrophic testes after torsion is lackMATERIALS AND METHODS ing because a biopsy of testes after orchidopexy is not done on both practical and ethical grounds. Adult 0150 days) and prepubertal (35 days) Sprague The exact mechanism of testicular injury due to torsion Dawley rats were acquired from University vivarium sources remains a topic of investigation. Possibilities include cellular and were maintained on a 12 hr:12 hr:light:dark cycle with hypoxia during the torsion period and injury resulting from ad libitum food and water. reperfusion.8 In experimental models the degree and duraBlood flow measurement by laser-Dopplerflowmetry. Rats tion of torsion can be set, and blood flow can be monitored. In were anesthetized with urethane (1mg./gm. body weight) earlier studies from this laboratory, 720" rotation of the testis and were placed in the supine position on a heating pad. A was observed to eliminate testicular blood flow during the right scrotal incision was made and the right testis was torsion period; nevertheless, blood flow returned to levels not delivered into the operative field. The gubemaculum was SUDificantly different from controls by 24 hours after torsion divided and the epididymis was freed from the testis to the r e ~ a i r . ~This - 8 was true in all cases except for testes that level of the Mum. Care was taken not to inadvertently injure were clearly infarcted. No information exists regarding the the testicular artery and veins. The testis was placed in a Pattern of blood flow return in the immediate p0St-torS.m specially fabricated testicle receptacle and was covered with Period; however, and this is a most critical time since SW@C@ water equilibrated mineral oil (fig. 1).The receptacle temperdecisions are being made based on whether or not the testm a t u e was monitored with a needle thermister probe and reperfuses soon after the torsion. The present study was maintained at 35C (fig. I).A rectal thermometer probe was designed to examine testidar microvascular blood flow be- inserted to insure that core body temperature was maintained at 37C. .fcwpted for publication November 19,1996. A laser-Doppler flowmeter ( m - 5 1 , Transonic Systems, for reprints: De artment of Urolo Box 422, univer- Ithaca, NY) with a Type N flow probe (Transonic SyssitY of irguua School of M&cine, Charlottes% VA 22908. tems, Ithaca, NY) wm used to determine microvascularblood by NIH grant DK 46179. 1493
1494
TESTICULAR BLOOD FLOW
FIG. 1. Temperature regulated (35C)testis receptacle with needle thermister probe (TP) and laser-Doppler flow probe ( F P ) held in position by micromanipulator. Anesthetized animal is prone, and leR testis is visible under mineral oil in testis receptacle.
flow. The probe was positioned with a micromanipulator (fig. 1) and by viewing through a dissecting microscope was adjusted to be approximately 0.5 mm. above the testicular surface in an area devoid of obvious arterioles and venules. Laser-Doppler flowmetry has been described elsewhere.Y.10.l 1 Briefly, a low intensity beam of monochromatic light, emitted from a laser diode inside the flowmeter, travels via the probe's fiber optic light guide through the probe head (1 mm. dia.) to illuminate the tissue under study. The laser beam is scattered by reflective components within the tissue to a depth of approximately 1 mm. A portion of the light is reflected back via the probe's receiving fiber optic light guide into a photo detector inside the flowmeter. The received light is reflected by stationary structures within the tissue a s well as by moving particles (red blood cells) which impart a frequency shift due to the Doppler effect. The received signal is processed by the ALF-21 and output is reported in perfusion units (PU) with one volt of output equalling 10 PU.9 Background values were determined over nonperfusing tissue five min. after the death of each animal and this value was subtracted from all values reported by the flowmeter. A computer interface and Flow trace software (Transonic Systems, Inc.. Ithaca, NY)was used to analyze the signal output. The software provides a scrolling display of flow and allows for determination of instantaneous or mean flow data and for measurement of the amplitude and frequency of testicular vasomotion.'O.l* Blood flow at different points on the testicular surface. Average microvascular blood flow was determined in each of four quadrants of five testes from five rats. Typical probe sites are illustrated in fig. 2. Each value recorded for each quadrant was the result of a 60 second rolling average. After the first observation for all quadrants had been recorded, the process was repeated two more times. The probe was repositioned from quadrant-to-quadrant between each reading. The three values within each quadrant were averaged to obtain the overall value for each quadrant in each testis. The effect of temperature on testicular microvascular blood floio. Six adult rats and five prepubertal rats were assigned to this portion of the study. The right testis was delivered and was placed in the testicle receptacle as previously described. The temperature of the mineral oil bath was initially set a t 32C. A 60 second average reading was taken after positioning the probe a s described above. The temperature was then increased and a reading was taken a t 35C and again a t 37C.
FIG. 2. Flow probe placement in quadrants of rat testis. Position of numbers indicates typical points within quadrants investigator would select for monitoring of blood flow. Flow probe diameter of one millimeter can be estimated from scale included in illustration.
The testis were allowed t o equilibrate for 10 min. at each temperature prior to taking readings. The flow probe was not moved between consecutive readings. Core body temperature was maintained by 37C. The effect of torsion on testicular blood flow. Rats were divided into three adult groups and three prepubertal groups (n = 8 or 9 each). The animals were prepared a s described above, but small bilateral scrota1 incisions were made to allow a window to the testis while still in the scrotum. Mean blood flow rate was determined from 60 second averages from both ipsilateral and contralateral testes while still in the scrotum. The right testis was then delivered to the testicle receptacle and prepared as previously described. Core body temperature was maintained at 37C and the bath temperature was maintained at 35C. Microvascular blood flow was again monitored prior to inducing experimental torsion. When blood flow was noted to be pulsatile in nature, amplitude in PU and frequency in cycles/min. were also recorded. Torsion was then induced by rotating the testis 720" along the longitudinal axis on the spermatic pedicle. The testis was then returned to the testis receptable and covered with mineral oil as before to protect against dehydration and to maintain appropriate testicular temperature. Care was taken to insure that the proper degree of torsion was maintained throughout the torsion period. Five minutes after induction of torsion testicular microvascular blood flow (PU) was determined. One, two, or four hours later, the torsion was relieved by counter-rotation and was replaced in the testis receptacle as before. Blood flow (60 second rolling averages) were determined a t 15, 30, and 60 mins. after relief of torsion. Finally, the adductor muscles of the thigh were exposed through a 5 mm incision and average muscle blood flow was also determined with the flow probe. The animals were sacrificed with an intracardiac injection of saturated KCL and background values were determined as described above. Statistics. All data were subjected to Chauvenet's criterion for homogeneity. All multiple comparisons were performed with analysis of variance followed by Tukey's range test (p c0.05). Comparison between adult and prepubertal blood
1495
TESTICULAR BLOOD FLOW
flow values within the temperature study was done by Student's t test. RESULTS
Microvascular perfusion of the rat testis. Initial microvascular blood flow in adult rat testes was 7.5 2 0.6 PU (mean 2 S.E.) in the scrotum and 5.9 5 0.5 PU in the testicle receptacle. The blood flow pattern initially observed in the testes of approximately 30% of adult rats was pulsatile, i.e. exhibited vasomotion, with a measurable frequency and amplitude (fig. 3). During the course of these and subsequent experiments it became evident that most rat testes experience periods with and without vasomotion; thus, many more of these testes would have exhibited vasomotion had we observed the testes over longer periods of time. Testes not evidencing vasomotion did not have different mean flow values from those testes which did. Blood flow at different points on the testicular surface. Averaged microvascular flow in the four quadrants of the adult rat testis were 8.30 2 0.47 PU, 5.94 2 0.79 PU, 8.01 2 0.60 PU, and 5.87 5 0.48 PU for quadrants 1, 2, 3, and 4, respectively. No significant differences existed among these values; however, if the data from quadrants 1and 3 (proximal pole) and quadrants 2 and 4 (distal pole) are pooled and analyzed by t-test the flow valves in the two regions are significantly different (p <0.05). In all subsequent studies flow probe placement was a t mid-testis rather than at the proximal or distal poles. Testicular development in prepubertal and adult rats. Histology of testes from the adult and prepubertal groups of rats confirmed the development status of the animals (fig. 4). Seminiferous tubules in prepubertal testes did not exhibit complete spermatogenesis (fig. 4, A) while seminiferous tubules in adult testes did exhibit complete spermatogenesis (fig. 4, B ) . Effect of temperature on testicular blood flow.Increasing FIG. 4. Histology of pre ubertal (A) and adult (Bf rat testes used ksticular temperature had no statistically significant effect in this study. Prepubertaftestes were still developing and did not on testicular blood flow in adult or prepubertal animals, but have tubules exhibiting complete spermatogenesis. Magnification microvascular flow in the prepubertal testis was significantly x200. higher than in the adult testes at every temperature studied (table 1). TABLE1 . Effect of testicular temperature on mean -t S.E. Effect of torsion on testicular blood flow. Testicular perfu- microvascular blood flow (P.U.)in prepubertal and adult rat testis sion data is detailed in tables 2 and 3 for adult and prepuTemperature Prepubertal Adult bertal animals, respectively. The data are also presented in 24.32 2 4.76' 6.92 z 1.5Eb 32C graphic form (fig. 5) to better illustrate the pattern of blood 29.20 2 5.66' 10.11 2r 2.7Eb 35c flow over time. 37c 26.54 2 5.13" 8.22 2 1.52b Adult animals. Five minutes after induction of 720" tora.b Means sharing the same superscript are not significantly different. sion, microvascular flow fell from a control value of 5.7 -+ 0.8 pu to 0.5 2 0.3 PU (table 2). Animals subjected to 1hr., 720" torsion had reperfusion values statistically indisguishable TABLE2. Laser-Doppler detection of microvascular perfusion from pre-torsion values within 15 mins. after torsion repair, (Perfusion Units, mean 5 S.E.) of adult rut testis before, during, but testes subjected to 2 and 4 hr. torsion required a t least and after 720"testicular torsion 30 mins. to attain values not significantly different from I
I
~~~~i~~ of Torsion (hr.)
cont,,,lt Flow
Torsion
15
1 2 4
5.7 ? 0.8 5.9 2 0.7 6.0 2 1.2
0.5 2 0.3 0.1 2 0.1 0.2 ? 0.1
4.0 2 1.0 1.9 2 0.4 1.6 2r 0.9
Time A&er Torsion Repair (min.) 30
60
1.5 3.4 2 0.7 3.8 2r 0.8
7.9 2 1.3
5.7
2
3.8 2 0.8 4.0 z 1.4
t Flow prior to torsion.
* Flow five min after induction of torsion.
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Ampmuds-397,092 ~ ~ " e =nt i ~36yt
PUfUSImUnlb
o 57
cywmn
0 0
15
30
Tim (sa)
FIG..3. Microvascular perfusion in adult rat testis exhibiting !asomotion. Flow values were obtained with testis in 35c testicle receptacle, and initial readings demonstrated vasomotion illustrated In approximately one-third of testes studied.
pre-torsion values (fig.5,A, arrows). By 1 hr. after repair of torsion, the testes subjected to 1 hr. torsion had flow values that were 1388 of pre-torsion values, demonstrating a tendency toward hyperemia. Reperfusion flow in testes experiencing 2 and 4 hr. torsion were only approximately 65% of pre-torsion values by 1 hr. after torsion (fig. 5, A). Overall blood flow, considering all the data for each group within the entire one hour of the study, was sianificantly less in testes
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TESTICULAR BLOOD FLOW
TABLE3. Laser-Doppkr detection of micivvasculur perfusion me^^ 2 S.E.)of the prepubertal mt testis before. dwinrr. and &r 720" testicular torsion
1
( l b f u a h Units,
1 2 4
14.3 2 1.3 14.0 2 2.1 12.7 f 1.8
1.2 2 0.4 0.6? 0.3 1.1 ? 0.3
15.1 2 2.9 10.5 t 1.9 8.2 t 1.5
10.5 2 3.2 7.2 2 1.5 5.8 5 1.5
10
14.52 2.9 12.8 t 2.1 7.4? 1.1
t Flow prim to torsion.
O J
Flow five min after induction of torsion.
15
I
30
Tim (m.1
A.
Adult
-
FIG. 6. Microvascular perfusion in adult rat testis 60 min. after
Torsion Time I hr t .
2hr 41K
repair of 1 hr. torsion. Vasomotion did not return after torsion repair.
turned to normal (fig. 5, B). One hour after torsion repair microvascular flow values were 102%, 91%, and 59% of initial flow values in testes subjected to 1 hr., 2 hr., and 4 hr. torsion, respectively. Total flow during the entire 60 min. reperfusion period in this study was significantly lower in animals subjected to 4 hr. torsion than those subjected to 1or 2 hr. torsion (fig. 5, B). In prepubertal rat testes before torsion testidar microvascular blood flow was similar to flow values obtained from thigh muscle, and thigh muscle values were not significantly different between adult and prepubertal animals. Prepubertal thigh muscle microvasculature did not exhibit vasomotion, and no prepubertal testis exhibited vasomotion after torsion repair. In no case was contralateral blood flow significantly affected by ipsilateral torsion (not shown).
(i muscle blood flow) -___--___
A
m
c I
ij 10 T
m
"1
B.
Prepubertal T
DISCUSSION
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'
,U ,
c o
,
,
l 15
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,
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l
l
30
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,
,
~
60 4
Time after torsion repair (rnin)
FIG. 5. Microvascular perfusion (mean t S.E.) in adult (A) and prepubertal ( B ) rat testes before and after 1,2,or 4 hr. 720' testicular torsion. Increasing torsion periods decrease re rfusion flow within times studied. Arrows indicate time beyond wEch microvascular flow values had returned and remained not Significantly different from pre-torsionvalues. a*bM e r e n t letters on the graph lines indicate that overall reperfusion data within entire 60 min. time period for torsion group are significantly different (p (0.05) from data of other torsion oups. C, control or pre-torsion blood flow. 0, flow values obtained %inp the period of torsion. with 2 and 4 hr. torsion than in testis with 1hr. torsion (fig. 5, A). Testicular microvascular flow in adult animals before and after torsion was less than that detected in thigh muscle (fig. 5, A). Vasomotion was never detected after torsion repair (fig. 6) and was not detected in thigh muscle. Prepubertal animals. Initial flow values obtained from testes in the testis receptacles were significantly higher than the comparable values in adult animals (table 2, table 3). Animals exhibiting vasomotion did so with a frequency of 12.2 2 0.52 cycledmin. and an amplitude of 4.07 2 0.95 PU. These values were not significantly different from the vasomotion parameters of adult animals (fig. 3). Experimental torsion reduced testicular microvascular perfusion to essentially zero (fig. 5 , B ) . In testes subjected to 1 or 2 hr. torsion reperfusion flow had returned to values not significantly different from pre-torsion values by 15 mins. after torsion repair (fig. 5,B , arrows). Testes subjected to 4 hr. torsion did not exhibit a time point beyond which blood flow had re-
The qualitative measurement of testicular blood flow with laser-Doppler flowmetry provides advantages over previous measurement techniques. Most importantly, this method allows continuous measurement of microvascular blood flow whereas many previous techniques had allowed only static determination of flow at predetermined time points. Changes in vascular microcirculation and microvascular permeability can conceivably have profound effects on testicular function. Inflow of peptide hormones, substrates, oxygen, and other factors as well as outflow of testosterone are related to microvascular blood flow and potentially regulated by it. Microvascular p e h i o n did not vary significantly between different quadrants of the rat testis; however, pooled results within the proximal and distal poles of the testis did reveal a significant difference (p <0.05) between poles. The meaning of this is uncertain and deserves further study; however, in all the subsequent experiments the flow probe was consistently placed at mid-testis to eliminate pole effects. Clearly, the present data reflect microvascular perfusion around seminiferous tubules only at the testicular surface. Preliminary experiments not detailed here showed that microvascular perfusion valves were not changed by removal of the tunia albuginea over the tubules monitored nor by probing tubules somewhat deeper (2-3 mm.) in the tissue. Thus, while the present technique cannot monitor perfusion in deep microvascular beds, we believe the surface microvasculature do reflect an activity that is similar throughout the testis. It is well established that cryptorchidism or exposing the testis to abdominal temperatures is deleterious to testis function.12sl3 Consistent with previous reports using different techniques, the present results demonstrate a lack of effectof physiological temperature increases on testicular blood flow14.l5 thus indicating that alteration in microvascufa blood flow is not a part of the mechanism by W h C h temperature-related testicular damage occurs. Adult and prepubertal animals commonly exhibited CYChcal variation in blood flow, or vasomotion (fig.3). This Ph*
1497
TESTICULAR BLOOD FLOW
nomenon has been observed in several tissues16 including rat kstes.'O. 11 These oscillations represent local changes in blood flow," and various theories have been proposed concerning the mechanism behind the phenomenon. These include microvascular smooth muscle activity, contraction of the testicular capsule, and interference with testicular venous outflow. The physiological consequence of vasomotion is &o largely unknown, but several studies have suggested a relationship to transvascular fluid exchange.17.18Damber et a1.10.11 have found vasomotion in all control rats and reported that vasomotion is established by 24 days of age.19 Subsequent to the completion of these experiments it was determined that most animals not exhibiting vasomotion upon initial examination will initiate vasomotion if observed for several minutes. Currently, 80%-90% of control animals in our laboratory exhibit vasomotion within 30 min. of initial exposure of the testis. The present data also confirm that prepubertal rats do exhibit testicular vasomotion by 35 days of age. A presumably correlated fact is that testosterone concentrations play an important role in the development of testicular vasomotion.20 Blood flow values in prepubertal animals were significantly higher than those recorded in adults. The physiological meaning of this is unclear. The prepubertal testis is actively growing and certainly has a high metabolic demand while adult testis growth has essentially ceased. Nevertheless, the adult testis also has a high metabolic demand for the support of spermatogenesis. Also, seminiferous tubule diameters are smaller in prepubertal than adult animals (fig. 4). This means more tubules and their vasculature can potentially be monitored within the 1 mm effective depth of the probe, and the higher blood flow recorded for prepubertal testes could be reflective of a higher capillarity per unit volume of testis rather than a higher flow through each unit of capillary. While the microvascular blood flow differences detected in the present study require further investigation; it should be noted that Damber, et a1.20 did not note any differences in flow parameters between adult and prepubertal animals. Interestingly, amplitude and frequency of vasomotor activity in prepubertal and adult animals were roughly equal despite the differences in average flow. It seems likely that once a developing testis receives the appropriate signals or conditions to exhibit vasomotion, the vasomotion proceeds with adult characteristics. Previous studies from this laboratory have demonstrated that 720" rotation of the rat testis virtually eliminates blood flow during the torsion period.3.8 This was verified in the Present study using a different technique. Microvascular flow in both adult and prepubertal animals fell to near zero immediately after torsion was induced (fig. 5). Return of testicd a r blood flow was significantly retarded in adult animals with torsion of 2 and 4 hrs. (fig. 5, A) and in prepubertal animals with torsion of 4 hrs. (fig. 5, B ) . It has previously been demonstrated in adult animals that total testicular blood flow returns to pre-torsion values within 24 hrs. even with torsions persisting for 4 hrs.;S thus, the present, more acute, changes detected in both adult and prepubertal testes might well vanish within 24 hrs. While the reduction in reperfusion flow with increasing times of torsion is resolved within 24 hours, the short-term changes shown here could well have an impact on post-torsion testicular damage3 Increasing times of torsion potentially: 1) increase h ~ o In~ JUrY as opposed to the reperfusion injury, 2) increase 1nJuryto nerneS innervating the vasculature and testicdar capsule, and 3) increase cell products leading to apoptosis of germ cells or somatic cells. Which of these have an impact on the microVasculature and the delayed return of microvascular flow after repair of testicular torsion remains to be determined.
Vasomotor activity after torsion repair (fig. 6) was always bsent. The reasons for this are unknown, but if vasomotion 3 required for appropriate delivery of metabolites, oxygen, or ell signalling molecules as has been speculated, then its lbsence could play a role in the permanent loss of spermatlgenesis experienced after significant periods of torsion. Vhether or not vasomotion ever returns to the post-torsion estis remains unknown. Contralateral blood flow was unaffected by ipsilateral tor;ion in both adult and prepubertal animals. This result is in igreement with previous findings from this laboratory3. and ;upports the studies from this and other laboratories which lave found no contralateral effect from ipsilateral torsion.
CONCLUSIONS
Microvascular blood flow during the acute phase of post1torsion reperfusion is retarded by increasing periods of torsion in both adult and prepubertal testes. Nevertheless, there are differences in reperfusion flow between the two types of testes. In general, these differences are: 1) mean microvascular perfusion per unit of perenchymal volume is higher in prepubertal testes than in adult testes (table 1,fig. 51, and 2) the prepubertal testis demonstrates a more robust return to pre-torsion flow values after 2 hrs. torsion than does the adult testis (fig. 5). This implies that the prepubertal testis might be more refractory to acute periods of torsion than the adult testis, a result we have previously found in a study of testicular endocrine and exocrine function after torsion.Z1 Additionally in neither prepubertal nor adult testes did vasomotion return after repair of torsion. Future investigations will determine if the loss of vasomotion after torsion is permanent. It will also be important to understand the basic regulation of vasomotion, and to determine its physiological consequences.
REFERENCES
1. Prater, M. M. and Overdorf, B. S.: Testicular torsion: a surgical emergency. Am. Fam. Physician, 44:3, 1991. 2. Workman, S.J. and Kogan, B. A.: Old and new aspects of testicular torsion. Sem. Urol., 6 146, 1988. 3. Turner, T. T.: Acute experimental torsion: no effect on the contralateral testis. J. Androl., 6 65, 1984. 4. Oettle, A. G.and Hamson, R. G.: The histological changes produced in the rat testis by temporary and permanent occlusion of the testicular artery. J. Pathol. Bateriol., 64.273, 1954. 5. Cass, A. S., Cass, B. B. and Veeraraghavan, K.: Immediate exploration of the unilateral acute scrotum in young male subjects. J. Urol., 121: 829, 1980. 6. Bartsch, G.,Frank, S. and Marberger, H.:Testicular torsion: late results with special regard to fertility and endocrine function. J. Urol., 124 375, 1980. 7. Anderson, J. B. and Williamson, R. C. N.: The fate of the human testes following unilateral torsion of the spermatic cord. Br. J. Urol., 506. 698,1986. 8. Turner, T. T. and Brown, K. J.: Spermatic cord torsion: loss of spermatogenesis despite return of blood flow. Biol. Reprod., 4 9 401, 1993. 9. Bonner, R. F., Clem, T. R., Bowen, P. D. and Bowman, R. L.: Laser doppler continuous real-time monitor of pulsatile and mean blood flow in tissue micro-circulation. In: Scattering Techniques, Applied to Supra-Molecular and Nonequilibrium Systems. Edited by Chen, S. H., Chu, B. and Nossal, R. New York: Plenum, pp. 685, 1981. c 10. Damber, J. E., Lindahl, 0..Selstam, G. and Tenland, T.: Testicular blood flow measured with a laser Doppler flowmeter: acute effects of catecholamines. Acta Physiol. Scand., 115 209, 1982. 11. Damber, J. E., Lindahl, O., Selstam, G. and Tenland, T.: Rhythmical oscillations in rat testicular microcirculation as recorded by laser Doppler flowmetry. Acta Physiol. Scand., 118: 117, 1983.
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TESTICULAR BLOOD FLOW
12. Nakamura, M., Hall, P. F. and &to, J.: Temperature effect on cell-free protein synthesis of the rat testis. F.E.B.S. Letters, 112: 221, 1980. 13. Galil, K. A. A. and Setchell, B. P.: Effects of local heating of the testes on the concentration of testosterone in jugular and testicular venous blood of rats and on testosterone production in vitro. Int. J. Androl., 11: 61, 1988. 14. Glover, T. D.: The influence of temperature on flow of blood in the testis and scrotum of rats. Proc. Roy. SOC.Med., 5 9 765, 1966. 15. Setchell, B. P., Waites, G. M. H. and Thornburg, G. D.: Blood flow in the testis of the conscious ram measured with krypton85. Circ. Res., 1 8 755, 1966. 16. Funk. W. and Intaglietta, M.: Spontaneous arteriolar vasomotion. Progress in Applied Microcirculation, 3: 66, 1983.
17. Intaglietta, M.: Arteriolar vasomotion: normal physiological activity or defense mechanism. Diab. and Metab., 2 4 489, 1988. 18. Widmark, A,, Damber, J. E. and Bergh, A.: Relationship between human chorionic gonadotropin induced changes in testicular microcirculation and the formation of testicular interstitial fluid. J. Endocrinol., 109.419, 1986. 19. Damber, J. E., Bergh, A. and Widmark, A.: Age related differences in testicular microcirculation. Int. J. Androl.. 13: 197, 1990. 20. Collin, 0.. Bergh, A,, Damber, J. E. and Widmark. A.: Control of testicular vasomotion by testosterone and tubular factors in rats. J. Reprod. Fert., 97: 115, 1993. 21. Becker, E. J. and Turner, T. T.: Endocrine and exocrine effi.cts of testicular torsion in the prepubertal and adult rat. J. Androl., 1 6 342, 1995.
THE JOURNAL
Vol. 167. 1493-1498, April 1997 Printed in U S A .
OF UROLOCY
Copyright 0 1997 by h m c m U R O ~ I CASSOCIATION, AL hc.
MICROVASCULAR BLOOD FLOW IS ALTERED AFTER REPAIR OF TESTICULAR TORSION IN THE RAT E. J. BECKER, H. M. PRILLAMAN AND T. T. TURNER* From the Department of Urology and Department of Cell Biology, University of Virginia School of Medicine, Charlottesville, Virginia
ABSTRACT
Purpose: Previous studies have shown that experimental testicular torsion with a duration of 1hr. or longer causes irreversible damage to the rat testis, but that testicular blood flow values are normal 24 hrs. after repair of torsion. More acute evaluation of return blood flow after repair of torsion has not been performed and was the topic of this study. Materials and Methods: Laser-Doppler flowmetry was used to evaluate teaticular microvascular blood flow before application of 1,2, or 4 hr., 720" torsion, during torsion, and at several time points after repair of torsion. Experiments were performed in both adult and prepubertal rats. Results: Testicular torsion essentially eliminated blood flow in both adult and prepubertal testes. Considering all the flow data within each group after torsion repair, increasing time of torsion was associated with significantly less return blood flow in both adult and prepubertal animals. Interestingly, only the four hour torsion data was associated with reduced return flow in prepubertal animals while both two and four hour torsion were associated with poor return flow in adult animals. Vasomotion, or pulsatile microvascular flow, often seen before torsion in both adult and prepubertal animals, was never seen after torsion repair. Conclusions: Increasing times of torsion are associated with lower microvascular blood flow values during the hour following the relief of torsion. Vasomotion is eliminated by torsion during the period studied. Whether vasomotion returns is unknown, but altering this flow pattern might be involved in the mechanism of injury caused by acute torsion. KEY WORDS:micmvasculature, blood flow,testis, rat, vasomotion Testicular torsion is defined as a twisting of the spermatic fore, during, and immediately (5-60 minutes) after various cord which results in impedance of blood flow to the involved durations of torsion. Testicular torsion primarily affecta pen-pubertal males alorgan.l.2 The degree and duration of torsion in the clinical setting is widely variable. Work with various animal models though most of the research published concerning torsion has has demonstrated a clear relationship between duration of involved adult animal models. The prepubertal testis is still in terms of size, vascular architecture, and the torsion and the resultant damage to the torsed t e s t i ~ . ~developing .~ Conventional operative management calls for detorsion of complex cellular relationships which allow for the completion the testis and observation; testes which reperfuse are left in of spermatogenesis. Since the vascular consequences of tesplace and are pexed to the scrotal septum. Testes which ticular torsion might be different in prepubertal and adult appear infarcted are removed. The exact incidence of later testes, we have performed the present studies in both types of atrophy in "salvaged" testes is unknown but by most reports animals. appears to be quite high.5,6,7 Clinical data concerning the relative function of these atrophic testes after torsion is lackMATERIALS AND METHODS ing because a biopsy of testes after orchidopexy is not done on both practical and ethical grounds. Adult 0150 days) and prepubertal (35 days) Sprague The exact mechanism of testicular injury due to torsion Dawley rats were acquired from University vivarium sources remains a topic of investigation. Possibilities include cellular and were maintained on a 12 hr:12 hr:light:dark cycle with hypoxia during the torsion period and injury resulting from ad libitum food and water. reperfusion.8 In experimental models the degree and duraBlood flow measurement by laser-Dopplerflowmetry. Rats tion of torsion can be set, and blood flow can be monitored. In were anesthetized with urethane (1mg./gm. body weight) earlier studies from this laboratory, 720" rotation of the testis and were placed in the supine position on a heating pad. A was observed to eliminate testicular blood flow during the right scrotal incision was made and the right testis was torsion period; nevertheless, blood flow returned to levels not delivered into the operative field. The gubemaculum was SUDificantly different from controls by 24 hours after torsion divided and the epididymis was freed from the testis to the r e ~ a i r . ~This - 8 was true in all cases except for testes that level of the Mum. Care was taken not to inadvertently injure were clearly infarcted. No information exists regarding the the testicular artery and veins. The testis was placed in a Pattern of blood flow return in the immediate p0St-torS.m specially fabricated testicle receptacle and was covered with Period; however, and this is a most critical time since SW@C@ water equilibrated mineral oil (fig. 1).The receptacle temperdecisions are being made based on whether or not the testm a t u e was monitored with a needle thermister probe and reperfuses soon after the torsion. The present study was maintained at 35C (fig. I).A rectal thermometer probe was designed to examine testidar microvascular blood flow be- inserted to insure that core body temperature was maintained at 37C. .fcwpted for publication November 19,1996. A laser-Doppler flowmeter ( m - 5 1 , Transonic Systems, for reprints: De artment of Urolo Box 422, univer- Ithaca, NY) with a Type N flow probe (Transonic SyssitY of irguua School of M&cine, Charlottes% VA 22908. tems, Ithaca, NY) wm used to determine microvascularblood by NIH grant DK 46179. 1493
1494
TESTICULAR BLOOD FLOW
FIG. 1. Temperature regulated (35C)testis receptacle with needle thermister probe (TP) and laser-Doppler flow probe ( F P ) held in position by micromanipulator. Anesthetized animal is prone, and leR testis is visible under mineral oil in testis receptacle.
flow. The probe was positioned with a micromanipulator (fig. 1) and by viewing through a dissecting microscope was adjusted to be approximately 0.5 mm. above the testicular surface in an area devoid of obvious arterioles and venules. Laser-Doppler flowmetry has been described elsewhere.Y.10.l 1 Briefly, a low intensity beam of monochromatic light, emitted from a laser diode inside the flowmeter, travels via the probe's fiber optic light guide through the probe head (1 mm. dia.) to illuminate the tissue under study. The laser beam is scattered by reflective components within the tissue to a depth of approximately 1 mm. A portion of the light is reflected back via the probe's receiving fiber optic light guide into a photo detector inside the flowmeter. The received light is reflected by stationary structures within the tissue a s well as by moving particles (red blood cells) which impart a frequency shift due to the Doppler effect. The received signal is processed by the ALF-21 and output is reported in perfusion units (PU) with one volt of output equalling 10 PU.9 Background values were determined over nonperfusing tissue five min. after the death of each animal and this value was subtracted from all values reported by the flowmeter. A computer interface and Flow trace software (Transonic Systems, Inc.. Ithaca, NY)was used to analyze the signal output. The software provides a scrolling display of flow and allows for determination of instantaneous or mean flow data and for measurement of the amplitude and frequency of testicular vasomotion.'O.l* Blood flow at different points on the testicular surface. Average microvascular blood flow was determined in each of four quadrants of five testes from five rats. Typical probe sites are illustrated in fig. 2. Each value recorded for each quadrant was the result of a 60 second rolling average. After the first observation for all quadrants had been recorded, the process was repeated two more times. The probe was repositioned from quadrant-to-quadrant between each reading. The three values within each quadrant were averaged to obtain the overall value for each quadrant in each testis. The effect of temperature on testicular microvascular blood floio. Six adult rats and five prepubertal rats were assigned to this portion of the study. The right testis was delivered and was placed in the testicle receptacle as previously described. The temperature of the mineral oil bath was initially set a t 32C. A 60 second average reading was taken after positioning the probe a s described above. The temperature was then increased and a reading was taken a t 35C and again a t 37C.
FIG. 2. Flow probe placement in quadrants of rat testis. Position of numbers indicates typical points within quadrants investigator would select for monitoring of blood flow. Flow probe diameter of one millimeter can be estimated from scale included in illustration.
The testis were allowed t o equilibrate for 10 min. at each temperature prior to taking readings. The flow probe was not moved between consecutive readings. Core body temperature was maintained by 37C. The effect of torsion on testicular blood flow. Rats were divided into three adult groups and three prepubertal groups (n = 8 or 9 each). The animals were prepared a s described above, but small bilateral scrota1 incisions were made to allow a window to the testis while still in the scrotum. Mean blood flow rate was determined from 60 second averages from both ipsilateral and contralateral testes while still in the scrotum. The right testis was then delivered to the testicle receptacle and prepared as previously described. Core body temperature was maintained at 37C and the bath temperature was maintained at 35C. Microvascular blood flow was again monitored prior to inducing experimental torsion. When blood flow was noted to be pulsatile in nature, amplitude in PU and frequency in cycles/min. were also recorded. Torsion was then induced by rotating the testis 720" along the longitudinal axis on the spermatic pedicle. The testis was then returned to the testis receptable and covered with mineral oil as before to protect against dehydration and to maintain appropriate testicular temperature. Care was taken to insure that the proper degree of torsion was maintained throughout the torsion period. Five minutes after induction of torsion testicular microvascular blood flow (PU) was determined. One, two, or four hours later, the torsion was relieved by counter-rotation and was replaced in the testis receptacle as before. Blood flow (60 second rolling averages) were determined a t 15, 30, and 60 mins. after relief of torsion. Finally, the adductor muscles of the thigh were exposed through a 5 mm incision and average muscle blood flow was also determined with the flow probe. The animals were sacrificed with an intracardiac injection of saturated KCL and background values were determined as described above. Statistics. All data were subjected to Chauvenet's criterion for homogeneity. All multiple comparisons were performed with analysis of variance followed by Tukey's range test (p c0.05). Comparison between adult and prepubertal blood
1495
TESTICULAR BLOOD FLOW
flow values within the temperature study was done by Student's t test. RESULTS
Microvascular perfusion of the rat testis. Initial microvascular blood flow in adult rat testes was 7.5 2 0.6 PU (mean 2 S.E.) in the scrotum and 5.9 5 0.5 PU in the testicle receptacle. The blood flow pattern initially observed in the testes of approximately 30% of adult rats was pulsatile, i.e. exhibited vasomotion, with a measurable frequency and amplitude (fig. 3). During the course of these and subsequent experiments it became evident that most rat testes experience periods with and without vasomotion; thus, many more of these testes would have exhibited vasomotion had we observed the testes over longer periods of time. Testes not evidencing vasomotion did not have different mean flow values from those testes which did. Blood flow at different points on the testicular surface. Averaged microvascular flow in the four quadrants of the adult rat testis were 8.30 2 0.47 PU, 5.94 2 0.79 PU, 8.01 2 0.60 PU, and 5.87 5 0.48 PU for quadrants 1, 2, 3, and 4, respectively. No significant differences existed among these values; however, if the data from quadrants 1and 3 (proximal pole) and quadrants 2 and 4 (distal pole) are pooled and analyzed by t-test the flow valves in the two regions are significantly different (p <0.05). In all subsequent studies flow probe placement was a t mid-testis rather than at the proximal or distal poles. Testicular development in prepubertal and adult rats. Histology of testes from the adult and prepubertal groups of rats confirmed the development status of the animals (fig. 4). Seminiferous tubules in prepubertal testes did not exhibit complete spermatogenesis (fig. 4, A) while seminiferous tubules in adult testes did exhibit complete spermatogenesis (fig. 4, B ) . Effect of temperature on testicular blood flow.Increasing FIG. 4. Histology of pre ubertal (A) and adult (Bf rat testes used ksticular temperature had no statistically significant effect in this study. Prepubertaftestes were still developing and did not on testicular blood flow in adult or prepubertal animals, but have tubules exhibiting complete spermatogenesis. Magnification microvascular flow in the prepubertal testis was significantly x200. higher than in the adult testes at every temperature studied (table 1). TABLE1 . Effect of testicular temperature on mean -t S.E. Effect of torsion on testicular blood flow. Testicular perfu- microvascular blood flow (P.U.)in prepubertal and adult rat testis sion data is detailed in tables 2 and 3 for adult and prepuTemperature Prepubertal Adult bertal animals, respectively. The data are also presented in 24.32 2 4.76' 6.92 z 1.5Eb 32C graphic form (fig. 5) to better illustrate the pattern of blood 29.20 2 5.66' 10.11 2r 2.7Eb 35c flow over time. 37c 26.54 2 5.13" 8.22 2 1.52b Adult animals. Five minutes after induction of 720" tora.b Means sharing the same superscript are not significantly different. sion, microvascular flow fell from a control value of 5.7 -+ 0.8 pu to 0.5 2 0.3 PU (table 2). Animals subjected to 1hr., 720" torsion had reperfusion values statistically indisguishable TABLE2. Laser-Doppler detection of microvascular perfusion from pre-torsion values within 15 mins. after torsion repair, (Perfusion Units, mean 5 S.E.) of adult rut testis before, during, but testes subjected to 2 and 4 hr. torsion required a t least and after 720"testicular torsion 30 mins. to attain values not significantly different from I
I
~~~~i~~ of Torsion (hr.)
cont,,,lt Flow
Torsion
15
1 2 4
5.7 ? 0.8 5.9 2 0.7 6.0 2 1.2
0.5 2 0.3 0.1 2 0.1 0.2 ? 0.1
4.0 2 1.0 1.9 2 0.4 1.6 2r 0.9
Time A&er Torsion Repair (min.) 30
60
1.5 3.4 2 0.7 3.8 2r 0.8
7.9 2 1.3
5.7
2
3.8 2 0.8 4.0 z 1.4
t Flow prior to torsion.
* Flow five min after induction of torsion.
SbodFbw=586t051 Peduawnwiltl
Ampmuds-397,092 ~ ~ " e =nt i ~36yt
PUfUSImUnlb
o 57
cywmn
0 0
15
30
Tim (sa)
FIG..3. Microvascular perfusion in adult rat testis exhibiting !asomotion. Flow values were obtained with testis in 35c testicle receptacle, and initial readings demonstrated vasomotion illustrated In approximately one-third of testes studied.
pre-torsion values (fig.5,A, arrows). By 1 hr. after repair of torsion, the testes subjected to 1 hr. torsion had flow values that were 1388 of pre-torsion values, demonstrating a tendency toward hyperemia. Reperfusion flow in testes experiencing 2 and 4 hr. torsion were only approximately 65% of pre-torsion values by 1 hr. after torsion (fig. 5, A). Overall blood flow, considering all the data for each group within the entire one hour of the study, was sianificantly less in testes
1496
TESTICULAR BLOOD FLOW
TABLE3. Laser-Doppkr detection of micivvasculur perfusion me^^ 2 S.E.)of the prepubertal mt testis before. dwinrr. and &r 720" testicular torsion
1
( l b f u a h Units,
1 2 4
14.3 2 1.3 14.0 2 2.1 12.7 f 1.8
1.2 2 0.4 0.6? 0.3 1.1 ? 0.3
15.1 2 2.9 10.5 t 1.9 8.2 t 1.5
10.5 2 3.2 7.2 2 1.5 5.8 5 1.5
10
14.52 2.9 12.8 t 2.1 7.4? 1.1
t Flow prim to torsion.
O J
Flow five min after induction of torsion.
15
I
30
Tim (m.1
A.
Adult
-
FIG. 6. Microvascular perfusion in adult rat testis 60 min. after
Torsion Time I hr t .
2hr 41K
repair of 1 hr. torsion. Vasomotion did not return after torsion repair.
turned to normal (fig. 5, B). One hour after torsion repair microvascular flow values were 102%, 91%, and 59% of initial flow values in testes subjected to 1 hr., 2 hr., and 4 hr. torsion, respectively. Total flow during the entire 60 min. reperfusion period in this study was significantly lower in animals subjected to 4 hr. torsion than those subjected to 1or 2 hr. torsion (fig. 5, B). In prepubertal rat testes before torsion testidar microvascular blood flow was similar to flow values obtained from thigh muscle, and thigh muscle values were not significantly different between adult and prepubertal animals. Prepubertal thigh muscle microvasculature did not exhibit vasomotion, and no prepubertal testis exhibited vasomotion after torsion repair. In no case was contralateral blood flow significantly affected by ipsilateral torsion (not shown).
(i muscle blood flow) -___--___
A
m
c I
ij 10 T
m
"1
B.
Prepubertal T
DISCUSSION
O
'
,U ,
c o
,
,
l 15
I
l
,
l
l
l
30
I
,
,
~
60 4
Time after torsion repair (rnin)
FIG. 5. Microvascular perfusion (mean t S.E.) in adult (A) and prepubertal ( B ) rat testes before and after 1,2,or 4 hr. 720' testicular torsion. Increasing torsion periods decrease re rfusion flow within times studied. Arrows indicate time beyond wEch microvascular flow values had returned and remained not Significantly different from pre-torsionvalues. a*bM e r e n t letters on the graph lines indicate that overall reperfusion data within entire 60 min. time period for torsion group are significantly different (p (0.05) from data of other torsion oups. C, control or pre-torsion blood flow. 0, flow values obtained %inp the period of torsion. with 2 and 4 hr. torsion than in testis with 1hr. torsion (fig. 5, A). Testicular microvascular flow in adult animals before and after torsion was less than that detected in thigh muscle (fig. 5, A). Vasomotion was never detected after torsion repair (fig. 6) and was not detected in thigh muscle. Prepubertal animals. Initial flow values obtained from testes in the testis receptacles were significantly higher than the comparable values in adult animals (table 2, table 3). Animals exhibiting vasomotion did so with a frequency of 12.2 2 0.52 cycledmin. and an amplitude of 4.07 2 0.95 PU. These values were not significantly different from the vasomotion parameters of adult animals (fig. 3). Experimental torsion reduced testicular microvascular perfusion to essentially zero (fig. 5 , B ) . In testes subjected to 1 or 2 hr. torsion reperfusion flow had returned to values not significantly different from pre-torsion values by 15 mins. after torsion repair (fig. 5,B , arrows). Testes subjected to 4 hr. torsion did not exhibit a time point beyond which blood flow had re-
The qualitative measurement of testicular blood flow with laser-Doppler flowmetry provides advantages over previous measurement techniques. Most importantly, this method allows continuous measurement of microvascular blood flow whereas many previous techniques had allowed only static determination of flow at predetermined time points. Changes in vascular microcirculation and microvascular permeability can conceivably have profound effects on testicular function. Inflow of peptide hormones, substrates, oxygen, and other factors as well as outflow of testosterone are related to microvascular blood flow and potentially regulated by it. Microvascular p e h i o n did not vary significantly between different quadrants of the rat testis; however, pooled results within the proximal and distal poles of the testis did reveal a significant difference (p <0.05) between poles. The meaning of this is uncertain and deserves further study; however, in all the subsequent experiments the flow probe was consistently placed at mid-testis to eliminate pole effects. Clearly, the present data reflect microvascular perfusion around seminiferous tubules only at the testicular surface. Preliminary experiments not detailed here showed that microvascular perfusion valves were not changed by removal of the tunia albuginea over the tubules monitored nor by probing tubules somewhat deeper (2-3 mm.) in the tissue. Thus, while the present technique cannot monitor perfusion in deep microvascular beds, we believe the surface microvasculature do reflect an activity that is similar throughout the testis. It is well established that cryptorchidism or exposing the testis to abdominal temperatures is deleterious to testis function.12sl3 Consistent with previous reports using different techniques, the present results demonstrate a lack of effectof physiological temperature increases on testicular blood flow14.l5 thus indicating that alteration in microvascufa blood flow is not a part of the mechanism by W h C h temperature-related testicular damage occurs. Adult and prepubertal animals commonly exhibited CYChcal variation in blood flow, or vasomotion (fig.3). This Ph*
1497
TESTICULAR BLOOD FLOW
nomenon has been observed in several tissues16 including rat kstes.'O. 11 These oscillations represent local changes in blood flow," and various theories have been proposed concerning the mechanism behind the phenomenon. These include microvascular smooth muscle activity, contraction of the testicular capsule, and interference with testicular venous outflow. The physiological consequence of vasomotion is &o largely unknown, but several studies have suggested a relationship to transvascular fluid exchange.17.18Damber et a1.10.11 have found vasomotion in all control rats and reported that vasomotion is established by 24 days of age.19 Subsequent to the completion of these experiments it was determined that most animals not exhibiting vasomotion upon initial examination will initiate vasomotion if observed for several minutes. Currently, 80%-90% of control animals in our laboratory exhibit vasomotion within 30 min. of initial exposure of the testis. The present data also confirm that prepubertal rats do exhibit testicular vasomotion by 35 days of age. A presumably correlated fact is that testosterone concentrations play an important role in the development of testicular vasomotion.20 Blood flow values in prepubertal animals were significantly higher than those recorded in adults. The physiological meaning of this is unclear. The prepubertal testis is actively growing and certainly has a high metabolic demand while adult testis growth has essentially ceased. Nevertheless, the adult testis also has a high metabolic demand for the support of spermatogenesis. Also, seminiferous tubule diameters are smaller in prepubertal than adult animals (fig. 4). This means more tubules and their vasculature can potentially be monitored within the 1 mm effective depth of the probe, and the higher blood flow recorded for prepubertal testes could be reflective of a higher capillarity per unit volume of testis rather than a higher flow through each unit of capillary. While the microvascular blood flow differences detected in the present study require further investigation; it should be noted that Damber, et a1.20 did not note any differences in flow parameters between adult and prepubertal animals. Interestingly, amplitude and frequency of vasomotor activity in prepubertal and adult animals were roughly equal despite the differences in average flow. It seems likely that once a developing testis receives the appropriate signals or conditions to exhibit vasomotion, the vasomotion proceeds with adult characteristics. Previous studies from this laboratory have demonstrated that 720" rotation of the rat testis virtually eliminates blood flow during the torsion period.3.8 This was verified in the Present study using a different technique. Microvascular flow in both adult and prepubertal animals fell to near zero immediately after torsion was induced (fig. 5). Return of testicd a r blood flow was significantly retarded in adult animals with torsion of 2 and 4 hrs. (fig. 5, A) and in prepubertal animals with torsion of 4 hrs. (fig. 5, B ) . It has previously been demonstrated in adult animals that total testicular blood flow returns to pre-torsion values within 24 hrs. even with torsions persisting for 4 hrs.;S thus, the present, more acute, changes detected in both adult and prepubertal testes might well vanish within 24 hrs. While the reduction in reperfusion flow with increasing times of torsion is resolved within 24 hours, the short-term changes shown here could well have an impact on post-torsion testicular damage3 Increasing times of torsion potentially: 1) increase h ~ o In~ JUrY as opposed to the reperfusion injury, 2) increase 1nJuryto nerneS innervating the vasculature and testicdar capsule, and 3) increase cell products leading to apoptosis of germ cells or somatic cells. Which of these have an impact on the microVasculature and the delayed return of microvascular flow after repair of testicular torsion remains to be determined.
Vasomotor activity after torsion repair (fig. 6) was always bsent. The reasons for this are unknown, but if vasomotion 3 required for appropriate delivery of metabolites, oxygen, or ell signalling molecules as has been speculated, then its lbsence could play a role in the permanent loss of spermatlgenesis experienced after significant periods of torsion. Vhether or not vasomotion ever returns to the post-torsion estis remains unknown. Contralateral blood flow was unaffected by ipsilateral tor;ion in both adult and prepubertal animals. This result is in igreement with previous findings from this laboratory3. and ;upports the studies from this and other laboratories which lave found no contralateral effect from ipsilateral torsion.
CONCLUSIONS
Microvascular blood flow during the acute phase of post1torsion reperfusion is retarded by increasing periods of torsion in both adult and prepubertal testes. Nevertheless, there are differences in reperfusion flow between the two types of testes. In general, these differences are: 1) mean microvascular perfusion per unit of perenchymal volume is higher in prepubertal testes than in adult testes (table 1,fig. 51, and 2) the prepubertal testis demonstrates a more robust return to pre-torsion flow values after 2 hrs. torsion than does the adult testis (fig. 5). This implies that the prepubertal testis might be more refractory to acute periods of torsion than the adult testis, a result we have previously found in a study of testicular endocrine and exocrine function after torsion.Z1 Additionally in neither prepubertal nor adult testes did vasomotion return after repair of torsion. Future investigations will determine if the loss of vasomotion after torsion is permanent. It will also be important to understand the basic regulation of vasomotion, and to determine its physiological consequences.
REFERENCES
1. Prater, M. M. and Overdorf, B. S.: Testicular torsion: a surgical emergency. Am. Fam. Physician, 44:3, 1991. 2. Workman, S.J. and Kogan, B. A.: Old and new aspects of testicular torsion. Sem. Urol., 6 146, 1988. 3. Turner, T. T.: Acute experimental torsion: no effect on the contralateral testis. J. Androl., 6 65, 1984. 4. Oettle, A. G.and Hamson, R. G.: The histological changes produced in the rat testis by temporary and permanent occlusion of the testicular artery. J. Pathol. Bateriol., 64.273, 1954. 5. Cass, A. S., Cass, B. B. and Veeraraghavan, K.: Immediate exploration of the unilateral acute scrotum in young male subjects. J. Urol., 121: 829, 1980. 6. Bartsch, G.,Frank, S. and Marberger, H.:Testicular torsion: late results with special regard to fertility and endocrine function. J. Urol., 124 375, 1980. 7. Anderson, J. B. and Williamson, R. C. N.: The fate of the human testes following unilateral torsion of the spermatic cord. Br. J. Urol., 506. 698,1986. 8. Turner, T. T. and Brown, K. J.: Spermatic cord torsion: loss of spermatogenesis despite return of blood flow. Biol. Reprod., 4 9 401, 1993. 9. Bonner, R. F., Clem, T. R., Bowen, P. D. and Bowman, R. L.: Laser doppler continuous real-time monitor of pulsatile and mean blood flow in tissue micro-circulation. In: Scattering Techniques, Applied to Supra-Molecular and Nonequilibrium Systems. Edited by Chen, S. H., Chu, B. and Nossal, R. New York: Plenum, pp. 685, 1981. c 10. Damber, J. E., Lindahl, 0..Selstam, G. and Tenland, T.: Testicular blood flow measured with a laser Doppler flowmeter: acute effects of catecholamines. Acta Physiol. Scand., 115 209, 1982. 11. Damber, J. E., Lindahl, O., Selstam, G. and Tenland, T.: Rhythmical oscillations in rat testicular microcirculation as recorded by laser Doppler flowmetry. Acta Physiol. Scand., 118: 117, 1983.
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TESTICULAR BLOOD FLOW
12. Nakamura, M., Hall, P. F. and &to, J.: Temperature effect on cell-free protein synthesis of the rat testis. F.E.B.S. Letters, 112: 221, 1980. 13. Galil, K. A. A. and Setchell, B. P.: Effects of local heating of the testes on the concentration of testosterone in jugular and testicular venous blood of rats and on testosterone production in vitro. Int. J. Androl., 11: 61, 1988. 14. Glover, T. D.: The influence of temperature on flow of blood in the testis and scrotum of rats. Proc. Roy. SOC.Med., 5 9 765, 1966. 15. Setchell, B. P., Waites, G. M. H. and Thornburg, G. D.: Blood flow in the testis of the conscious ram measured with krypton85. Circ. Res., 1 8 755, 1966. 16. Funk. W. and Intaglietta, M.: Spontaneous arteriolar vasomotion. Progress in Applied Microcirculation, 3: 66, 1983.
17. Intaglietta, M.: Arteriolar vasomotion: normal physiological activity or defense mechanism. Diab. and Metab., 2 4 489, 1988. 18. Widmark, A,, Damber, J. E. and Bergh, A.: Relationship between human chorionic gonadotropin induced changes in testicular microcirculation and the formation of testicular interstitial fluid. J. Endocrinol., 109.419, 1986. 19. Damber, J. E., Bergh, A. and Widmark, A.: Age related differences in testicular microcirculation. Int. J. Androl.. 13: 197, 1990. 20. Collin, 0.. Bergh, A,, Damber, J. E. and Widmark. A.: Control of testicular vasomotion by testosterone and tubular factors in rats. J. Reprod. Fert., 97: 115, 1993. 21. Becker, E. J. and Turner, T. T.: Endocrine and exocrine effi.cts of testicular torsion in the prepubertal and adult rat. J. Androl., 1 6 342, 1995.