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From the * Department of Urology and the
Department of Cell Biology, University of
Virginia Health Science System, Charlottesville, Virginia.
| Correspondence to: Dr Jeffrey J. Lysiak, Department of Urology Box 800422, University of Virginia Health System, Charlottesville, VA 22908 (e-mail: jl6n{at}virginia.edu ). |
| Received for publication October 3, 2001; accepted for publication January 7, 2002. |
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Abstract |
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Key words: Apoptosis, ischemia-reperfusion, oxygen radicals
Cell death is associated with IR of several organs, including the heart (Yin et al, 1997), kidney (Singbartl and Ley, 2000), and brain (Connolly et al, 1996), and the overall effect has been given the term "ischemia-reperfusion injury." IR injury in many aspects resembles an inflammatory response (Springer, 1995; Gute et al, 1998) characterized by an increase in neutrophils to the affected organ (Kelly et al, 1996) and an increase in oxidative stress (Fontana et al, 2001). Oxidative stress can arise by the generation of the superoxide anion (O2•-), hydrogen peroxide (H2O2), or the hydroxyl ion (HO•; Mates and Sanchez-Jimenez, 1999). Under nonpathologic conditions, the superoxide anion concentration is controlled by a family of superoxide dismutase (SOD) enzymes. SOD enzymes are located in the mitochondria, in the cytosol, or on extracellular membranes and may have either Cu, Fe, or Mn at their active site (Majima et al, 1998). SODs catalyze the dismutation of O2•- to H2O2 and O2 (Majima et al, 1998). Another enzyme, catalase, converts H2O2 to H2O and O2 (Lledias et al, 1998). The administration of SOD and catalase has proven beneficial in models of IR injury. Indeed, previous work from this laboratory has shown that the treatment of rats with SOD plus catalase prior to reperfusion of the testis and immediately thereafter provides partial rescue of spermatogenesis (Prillaman and Turner, 1997; Turner et al, 1997).
The aim of the present study was to further investigate the therapeutic
effects of SOD and catalase in IR of the rat testis in comparison to the
effects of a nonpeptidyl SOD mimic, M40403
(Salvemini et al, 1999). M40403 is derived from the macrocyclic ligand
1,4,7,10,13-pentaazacyclopentadecane, containing the added
bis-(cyclohexylpyridine) functionalities. M40403 is stable in whole rat blood
up to 10 hours and has been shown to be effective in inhibiting edema after
carrageenan injection into a rat paw and in reducing lipid peroxidation,
neutrophil infiltration, and the levels of tumor necrosis factor alpha
(TNF-
) and interleukin-1beta (IL-1ß) after splanchnic artery
occlusion in the rat (Salvemini et al,
1999). In the present study, acute testicular IR events such as
ROS generation and the recruitment of neutrophils to the testis were examined
as well as long-term effects on spermatogenesis. Understanding the role of ROS
in the pathology of testicular torsion and the effects of oxido-reductases may
lead to the design of new therapies for the protection of spermatogenesis as
well as to insights into other forms of oxidative stress injury.
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Materials and Methods |
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Assessment of Testicular Lipid Peroxidation
Testes were removed and decapsulated, and the resulting tissue—minus
the tunica albuginea, major testicular surface vessels, and rete
testis—was gently disrupted by extrusion through a 3-mL syringe hub. The
disrupted seminiferous tubules were centrifuged for 20 minutes at 13 000
x g at 4°C, and the supernatant testicular fluid was
collected. Lipid peroxidation products in the testicular fluid were assessed
by the TBARS method of Uchiyama and Mihara
(1978). This method detects
the formation of malonaldehyde (MDA), which arises from the peroxidation of
cell membrane lipids. MDA is measured by its stoichiometric binding to
thiobarbituric acid in heated acid solution and the reaction product's
subsequent fluorescence. Briefly, MDA standards were created by acid
hydrolysis of 1,1,3,3-tetraethoxypropane. MDA concentrations in the standards
were determined spectrophotometrically at 267 nm using a molar absorption
coefficient of 31 800 M-1 cm-1. Subsequently, 50-µL
portions of each testicular fluid sample and each standard were mixed with 300
µL of 1% H3PO4 in 0.1 N HCl and 100 µL of 0.6%
thiobarbituric acid in 0.1 N HCl. The mixture was heated at 100°C for 45
minutes, cooled on ice, and extracted with 600 µL of n-butanol.
The organic phase was separated by centrifugation. Fluorescence was measured
in a fluorescence spectrometer (LS-5B; Perkin-Elmer Ltd, Beacons Field,
Buckinghamshire, United Kingdom) with excitation of 532 nm and emission of 555
nm, and TBARS values were calculated and reported as MDA molar
concentrations.
Evaluation of Germ Cell Apoptosis
Germ cell apoptosis was examined immunohistochemically with the monoclonal
antibody F7-26 (Apostain, Alexis Corporation, San Diego, Calif) directed
against single-stranded DNA (ss-DNA). At the specified time points after
torsion repair, testes were removed from the scrotum, rinsed in saline,
immersed in Bouin fixative for 6 hours, and embedded in paraffin. The Apostain
technique was performed according to the manufacturer's protocol. Briefly,
sections were deparaffinized, rehydrated, rinsed in 5 mM MgCl2 in
phosphate-buffered saline (PBS), rinsed in deionized water (dH2O),
and incubated for 15 minutes in ice-cold 0.1 N HCl. Subsequently, sections
were rinsed in dH2O and incubated for 5 minutes in 5 mM
MgCl2, 0.2% Triton X-100 in PBS. The slides were then placed into
50-mL centrifuge tubes containing 30 mL of 50% formamide, and the tubes were
immersed for 20 minutes in 56°C water. After heating, the slides were
immediately removed and transferred into ice-cold PBS for 10 minutes. The
chilled slides were immersed in 3% H2O2 to block
endogenous peroxidases, immersed in 0.1% bovine serum albumin and 1% nonfat
dry milk to block nonspecific binding of antibody, rinsed in PBS, and
incubated overnight with a 1:100 dilution of F7-26. Slides were washed,
incubated for 1 hour with biotinylated rat anti-mouse antibody (Zymed, San
Francisco, Calif), and washed. The biotinylated secondary antibody was
visualized with avidin-biotin-peroxidase complex (Elite ABC Kit, Vector
Laboratories, Burlingame, Calif) and diaminobenzidine (Sigma Chemical Co, St.
Louis, Mo) as the chromogen. Sections were counterstained with hematoxylin,
dehydrated, and mounted. The number of apoptotic cells was evaluated by
counting the positively stained nuclei in 30 circular seminiferous tubule
cross sections per testis section. Data were averaged for each testis and
expressed as apoptotic nuclei per tubule cross section.
Experimental Testicular Torsion
Adult male Sprague-Dawley rats (450-550 g) were anesthetized with an
intraperitoneal injection of sodium pentobarbital (50 mg/kg), and the testis
was rotated as described by Turner et al
(1997). Briefly, the testis
was exteriorized through a low midline laparotomy, the gubernaculum was
divided, and the testis was freed from the epididymotesticular membrane. The
testis was then rotated 720° for 1 hour and placed in the abdomen with a
closed incision. At the appropriate time, the incision was reopened, the
testis was counterrotated to the natural position, the gubernaculum was
rejoined, and the testis was reinserted into the scrotum via the inguinal
canal. This treatment has been demonstrated to induce aspermatogenesis in the
affected testicle (Baker and Turner,
1995; Turner and Miller,
1997; Turner et al,
1997). At the time of repair, testes were examined and scored for
the apparent degree of ischemia and reperfusion, respectively. Testes were
collected either at 4 hours after the repair of torsion for the quantification
of neutrophils and lipid peroxidation or at 30 days after the repair of
torsion for the quantification of testis weight and daily sperm production
(DSP). Sham-operated animals were treated identically except that, upon
completion of the torsion maneuver, the testis was immediately
counterrotated.
Drug Infusions
Animals were divided into 6 groups: 1) sham torsion, or torsion plus
infusion, with 2) the vehicle (saline), 3) catalase (Sigma), 4) Cu-Zn SOD
(Sigma), 5) catalase plus SOD, and 6) M40403 (a generous gift from MetaPhore
Pharmaceuticals Inc, St Louis, Mo). Animals were subjected either to sham
operation or bilateral torsion as described above. All drugs were infused at a
concentration of 6 mg/kg of body weight using the procedure described by
Prillaman and Turner (1997).
Briefly, the right femoral vein was exposed, and a 30-gauge needle attached to
a 1-mL syringe via PE 10 tubing was inserted. The syringe was placed into an
infusion pump (Model 341A, Sage), and the drugs were infused at a rate of 0.67
mL/h. Infusions began 15 minutes prior to the repair of torsion and continued
for a total infusion time of 1.5 hours.
Evaluation of Testicular Neutrophil Content
Testicular neutrophil content was determined by myeloperoxidase (MPO)
assay. MPO is stored in primary granules of neutrophils, and the enzyme
activity is a common measure of neutrophil accumulation
(Grisham et al, 1990). Following testicular torsion and repair, testes were removed and snap frozen
at -80°C. Tissues were homogenized in 2.0 mL ice-cold 20 mM potassium
phosphate buffer, pH 7.4, and centrifuged at 17000 x g at
4°C for 30 minutes; the pellets were resuspended in ice-cold potassium
phosphate buffer, pH 7.4. Suspensions were then centrifuged 2 more times, and
0.5% (wt/vol) hexacyltrimethylammonium bromide, 10 mM EDTA in 50 mM potassium
phosphate buffer, pH 6.0, was added to the remaining pellet. Suspensions were
sonicated 5 times for 1 second each on ice, freezethawed 3 times, incubated at
4°C for 20 minutes, and centrifuged at 17000 x g for 15
minutes at 4°C. The protein concentration in the resulting supernatant was
determined using bicinchoninic acid assay (BCA Protein Assay, Pierce,
Rockford, Ill) to ensure that equal amounts of protein were assayed. Duplicate
samples of the supernatant were incubated with 0.2 mg/mL
o-dianisidine, 158 µM H2O2 in 50 mM
potassium phosphate buffer, pH 6.0, at a ratio of 4:1, and changes in
absorbance were detected at 450 nm using a microtiter photometric plate reader
(Titer-Tek, Huntsville, Ala). A series of preliminary experiments were
conducted to determine if catalase interfered with the MPO assay. In vitro
results revealed that concentrations of catalase estimated to be in the testis
during the infusion had no effect on the MPO assay. Higher concentrations of
catalase (>0.06 mg/mL in the assay reaction) did cause a
concentration-dependent decrease in the MPO assay (data not shown).
Evaluation of Testicular Subtunical Venule Neutrophil
Margination
The accumulations of neutrophils in the subtunical venules of the various
groups were examined 4 hours after the repair of torsion as well as from sham
controls. Briefly, hematoxylin and eosin-stained sections of testes were
examined using a Nikon FXA microscope with an attached digital camera
(CoolPIX, Nikon, Columbia, Md). Images were captured, and all subtunical
venules in a section were traced using ImagePro software (Molecular Dynamics
Inc, Sunnyvale, Calif). The intraluminal area of each vessel was determined.
Adherent neutrophils were then counted in 25-60 vessels from animals of each
group, and the numbers were expressed as neutrophils per square millimeter of
luminal area.
Evaluation of Sperm Production
Thirty days after the repair of testicular torsion or sham operation,
testes were removed and immediately weighed. DSP (sperm x
106/g/d) was also determined according to the method of Robb et al
(1978) as reported previously
from this laboratory (Lysiak et al,
2001). Briefly, testes were weighed, decapsulated, and homogenized
in 50 mL of 0.154 M NaCl, 0.5% Triton X-100 (vol/vol), 2% sodium azide, and
eosin Y. The concentration of condensed sperm nuclei was calculated using a
hemocytometer and expressed as DSP.
Statistical Analysis
Each data set was evaluated by Chauvenet's criterion for homogeneity
(Worthington and Jeffner,
1943). In some experiments, the issue was whether the data from
torsion testes were significantly different from the data from sham control
testes. In other experiments, the issue was whether treatment with a ROS
scavenger had a significant effect relative to the sham control group or,
depending on the experiment, to the group receiving torsion with saline
infusion. These control-vs-treatment evaluations were made with the Student's
t test (P <.05).
Further, in the antioxidant treatment experiments, it was of interest to determine what percentage of testes were "responders" to the various treatments with regard to the clinically relevant standards of testis weight and DSP. A responder within a given parameter was defined as a value more than 2 standard deviations higher than the mean of the group receiving torsion with saline infusion only.
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Results |
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Evaluation of Neutrophil Recruitment to Testicular Subtunical Venules
After Testicular Torsion
Testis sections from rats 4 hours after a sham operation revealed that very
few neutrophils adhered to the endothelium of subtunical venules
(Figure 3A and C), whereas
sections from rat testes 4 hours after the repair of testicular torsion with
saline infusion only displayed a significant increase in marginated
neutrophils in subtunical venules (Figure
3B and C). MPO activity increased significantly 4 hours after the
repair of torsion compared to MPO activity in sham-operated controls
(Figure 3D). This significant
increase in MPO activity is in agreement with the increase in subtunical
venule neutrophil counts and, taken together, the data indicate that
neutrophils are recruited to the testis as early as 4 hours after IR of the
rat testis.
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Testicular Lipid Peroxidation After Testicular Torsion
Testicular fluid isolated from testes 4 hours after the repair of
testicular torsion with saline infusion contained a significantly higher
concentration of MDA, the lipid peroxidation product, than testicular fluid
isolated from testes after sham operation
(Figure 4).
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Evaluation of the Testis 30 Days After the Repair of Testicular
Torsion
Thirty days after sham operation or after the repair of testicular torsion
with the animal receiving only a saline infusion, testes were assessed for
testicular weight and DSP. A significant decrease in testicular weight and DSP
occurred 30 days after the repair of testicular torsion with saline infusion
compared to sham-operated animals (Figure
5A and B).
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Reactive Oxygen Species Scavenger Treatments
Testicular MPO values determined 4 hours after repair of torsion with
saline infusion were significantly increased over MPO values of sham-operated
animals (Table 1). This
implication of recruitment of neutrophils to the testis was confirmed by
direct inspection of testicular venules. Neutrophil adhesion to testicular
venules (cells/mm2 vessel wall area) in testes 4 hours after the
repair of torsion with saline infusion (903 ± 159) was significantly
more than in sham-operated testes (187 ± 64). Intravenous infusion of
ROS scavengers at the time of torsion repair generally reduced MPO values
relative to those in testes after torsion repair not receiving ROS scavengers.
Infusion of catalase, SOD plus catalase, and M40403 all reduced MPO activity 4
hours after torsion repair to values not significantly different from
sham-operated values (Table 1).
Declines in MPO values were confirmed by decreases in neutrophil counts (data
not shown). TBARS values in animals that received catalase, SOD plus catalase,
and M40403 after torsion were also not significantly different from the TBARS
values of sham-operated animals (Table
1). Infusion of SOD alone provided no reduction in MPO and did not
reduce TBARS (Table 1). Thus,
catalase and SOD plus catalase returned mean MPO values completely to sham
control levels, and M40403 returned MPO values to approximately 73% of sham
controls. Infusion of catalase, SOD plus catalase, and M40403 all returned
mean TBARS values to 86%-98% of sham control values
(Table 1).
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Torsion without antioxidant infusion caused a significant reduction in testis weight and DSP (Figure 5; Table 2). Infusion with ROS scavengers at the time of torsion repair provided partial rescue of DSP 30 days after repair of torsion, though this rescue was far from complete (Table 2). Infusion with SOD, SOD plus catalase, and M40403 all provided a significant increase in DSP compared to DSP in animals that underwent torsion without antioxidant treatment (Table 2). These increases represented an approximate 25%-30% return of DSP. Further, 50% of the testes in each of these groups were classified as responders to treatment (>2 SD above the mean of torsion without antioxidant treatment) with regard to both testis weight and DSP (data not shown). Infusion of catalase at the time of torsion repair did not improve any measured parameter 30 days after torsion repair (Table 2).
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Discussion |
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Testicular torsion itself causes a significant increase in neutrophil adhesion to the testicular venous endothelium (Figure 2) and a corresponding increase in ROS, as assessed by the TBARS assay (Figure 3). It is well documented that activated neutrophils are potent generators of ROS (Pustovidko et al, 2000), and previous studies from this laboratory have demonstrated that the recruitment of neutrophils to the testis after torsion is essential for the observed pathology (Lysiak et al, 2001). Alleviation of oxidative stress has been shown beneficial in several models of disease such as myocardial infarction (McDonald et al, 1999), inflammation (Cuzzocrea et al, 2001), and stroke (Pluta et al, 2001). One strategy to reduce oxidative stress involves the elimination of ROS by ROS scavenger proteins. The administration of enzymes such as SOD and catalase can reduce the ROS burden and provide cellular protection from IR injury. Previous studies from this laboratory have demonstrated that the infusion of SOD plus catalase to rats 15 minutes prior to reperfusion of the testis and for 1 hour thereafter provided significant rescue of testis weight and DSP 30 days after IR of the testis (Prillaman and Turner, 1997). The therapy portion of the present study was designed to investigate the effects of SOD and catalase proteins independently, as well as the nonpeptide SOD mimetic M40403.
The infusion of animals with catalase, SOD plus catalase, or M40403 maintained MPO and TBARS values at levels not significantly different from sham controls in the acute period after torsion repair (Table 1). Interestingly, catalase alone was not among the treatments that provided protection for testis function when assessed 30 days after torsion repair, but SOD alone did provide protection (Table 2). Peltola et al (1992) reported that catalase activity in the adult rat testis (1-2 µg/mg protein) was much less than in liver (50-100 µg/mg protein) but that SOD activity in the testis (3-4 µg/mg protein) was approximately equivalent to that in liver. It might have been anticipated that a testis relatively deficient in catalase but with liver levels of SOD might benefit from infusion of catalase alone, since H2O2 would presumably build up because of the SOD activity dismuting O2•-. In fact, infusing catalase did reduce testicular oxidative stress when assessed 4 hours after the repair of torsion (Table 1), but the compound did not provide protection for DSP 30 days later (Table 2). On the other hand, SOD alone did not significantly suppress oxidative stress 4 hours after repair of torsion (Table 1) but did provide significant protection for DSP 30 days later (Table 2). The reasons for these contrasting effects are unclear but likely involve both the germ cells' relative sensitivity to the hydroxyl and superoxide radicals and the plasma half-times of SOD and catalase after infusion, which are different (Omar and McCord, 1990; Wengenact et al, 1997).
The observation that antioxidant treatments reduce neutrophil margination is not only mechanistically interesting, but it offers another possible explanation for the differential effects of SOD and catalase when administered alone. Numerous studies have demonstrated that prooxidant treatments, especially those increasing H2O2, induce the recruitment of neutrophils to the affected organ or cause adhesion of neutrophils to vascular endothelium (Ichikawa et al, 1997; Lefer et al, 1997; Terada et al, 1997; Okayama et al, 1998). It is possible that catalase was effective in reducing neutrophil recruitment and oxidative stress in the short term, but its relatively short half-life in plasma allowed a rise in H2O2 and subsequent recruitment of neutrophils even after the 4-hour posttorsion time point, and thus, the failure to protect spermatogenesis when assessed 30 days later. SOD administered alone might produce an acute rise in H2O2 that overwhelms native catalase in the short term but, in the long term, might continue to reduce tissue O2•- and to produce H2O2 at a lower level within the capacity of native catalase. This speculation would explain how catalase alone reduced oxidative stress when measured acutely but did not protect spermatogenesis in the long term and how SOD did not reduce oxidative stress when measured acutely but did provide significant protection of spermatogenesis in the long term.
Both the combination treatment with SOD plus catalase and the treatment with M40403 kept acute oxidative stress values statistically at the same level as sham controls (Table 1) and, importantly, provided significant protection for testis function 30 days later (Table 2). Interestingly, 50% of the testes in both of these groups were responders to treatment (ie, their DSP and testis weight values were >2 SD higher than those of the group receiving torsion without antioxidant treatment). Clearly, 50% of the testes in these groups did not respond to treatment, thus blunting the groups' mean responses and increasing data variability with each group. The reasons for the failure of some testes to respond are unknown but are likely related to the variability of blood flow return in the short term after torsion repair. The previous experience of this lab with observations of total organ blood flow (Turner and Brown, 1992) and microvascular blood flow (Lysiak et al, 2000a) after torsion repair is that blood flow return rates can vary by as much as 50% within the first 4 hours of reperfusion. This is a biological variable that cannot be controlled in either the experimental or clinical setting and will likely continue to be a factor affecting the success of medical therapies augmenting surgical repair of testicular torsion.
It is important to note that the nonpeptide SOD mimetic compound M40403 was
effective at providing significant protection to testicular spermatogenic
function (Table 2).
Immunogenicity of proteins like SOD and catalase themselves or even
recombinant SOD has proven problematic with regard to using these compounds in
therapeutic regimens for humans. As a result, attempts have been made to
produce molecules that are not immunogenic yet retain activity. M40403 is one
such compound. In a rat model of splanchnic artery occlusion and reperfusion,
M40403 significantly reduced neutrophil accumulation in the lung and ileum,
decreased plasma MDA concentrations, decreased the plasma levels of
TNF- and IL-1ß, and increased the mean survival
(Salvemini et al, 1999). Results from the present study confirm some of these earlier findings of the
efficacy of M40403 and suggest that the drug has a therapeutic role in IR
injury.
Finally, in the clinical setting, it typically cannot be determined in the short term if torsion repair has protected actual spermatogenesis. The reasons for this revolve around issues of patient age and the related ethical difficulties of obtaining a semen sample or testis biopsy. Thus, the clinical evaluation of the degree of damage to the testis resulting from the torsion episode is often limited to examination for testicular atrophy. At issue is the question, If testis size is within normal ranges after torsion repair, to what degree does spermatogenesis return to normal? Extending the analogy of experimental torsion in the rat to the human clinical condition and using testis weight as the homolog of the clinical estimation of testicular size, we have determined those testes that are responders to treatment based on testis weight and have determined the DSP values in those testes alone. As related previously, 50% of the testes in each of the SOD, SOD plus catalase, and M40403 infusion groups were responders to the treatments (Figure 6A). The mean DSP values for those testes (Figure 6B) are 69%, 58%, and 59% of the sham control value for the SOD, SOD plus catalase, and M40403 groups, respectively. Thus, on the basis of this experimental model, it would be expected that a testis responding to treatment based on testicular mass would have a 60%-70% salvage of spermatogenesis.
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Clearly, further examination of dosing regimens (drug concentrations, duration of administration, route of administration, etc) needs to be performed as well as consideration of other inhibitors of oxidative stress such as glutathione and glutathione peroxidase or inhibitors of xanthine oxidase and calcium transport. Such experiments are being undertaken by this laboratory as well as those examining further details on the molecular mechanisms involved in testicular IR injury.
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Acknowledgments |
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Footnotes |
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