The Correlation of Gene Expression of Inflammasome Indicators and Impaired Fertility in Rat Model of Spinal Cord Injury: A Time Course Study Banafsheh Nikmehr1, Mahshid Bazrafkan1, Gholamreza Hassanzadeh1, Abdolhossein Shahverdi2, Mohammad Ali Sadighi Gilani3, Sahar Kiani4, Tahmineh Mokhtari5, Farid Abolhassani1* Purpose: Expression assessment of the inflammasome genes in the acute and the chronic phases of Spinal cord injury (SCI) on adult rat testis and examination of associations between inflammasome complex expression and sperm parameters. Materials and methods: In this study, 25 adult male rats were randomly divided into 5 groups. SCI surgery was performed at T10-T11 level of rats’ spinal cord in four groups (SCI1, SCI3, SCI7, and SCI56). They were sac- rificed after 1day, 3days, 7days and 56 days post SCI, respectively. One group remained intact as control (Co). CASA analysis of sperm parameters and qRT-PCR (ASC and Caspase-1) were made in all cases. Results: Our data showed a severe reduction in sperm count and motility, especially on day 3 and 7. ASC gene expression had a non-significant increase on day 1 and 56 after surgery compared to control group. Caspase-1 expression increased significantly on day 3 post injury versus the control group (P = .009). Moreover, Caspase-1 overexpression, had significant correlations with sperm count (r = -0.555, P = .01) and sperm progressive motility (r = -0.524, P = .02). Conclusion: Inflammasome complex expression increase following SCI induction. This overexpression correlates to low sperm parameters in SCI rats. Keywords: spinal cord injury; infertility; testis, inflammasome; ASC; Caspase-1. INTRODUCTION Spinal cord injury (SCI) is a devastating clinical issue affecting 40 to 80 new cases per million population each year throughout the world and up to 90% of these cases are due to traumatic causes(1). According to a study conducted by a Specialized Spinal cord injury center in Tehran, Iran, the incidence of SCI is up to 2.36 per- sons per 10000 population with an average age of 29.1 years. SCI patients are at higher risk of morbidity and mortality because of complications related to the injury. Iran has younger SCI cases more than other developing countries and about 80% of whom are male. SCI peo- ple are usually at reproductive age, so fatherhood is a grave issue for this population(2). About 85% to 97% of SCI men suffer from impaired fertility caused by erec- tile dysfunction and ejaculatory problems(3). There are some assisted methods such as penile vibration (PVS) and electroejaculation (EEJ) to obtain the semen from these patients. However, most SCI men have a low se- men quality(4-6). Although testicular tissue becomes involved post-SCI, a limited number of studies have addressed this issue. Impaired spermatogenesis, vast germ cell apoptosis, inflammatory cytokines elevation, blood-testis barrier disruption and leukocytes influx have been demonstrat- ed as abnormal changes in testis after SCI inducing an inflammatory environment and unstable niche in this tissue(7-9). The inflammasome is a multi-protein complex that is a part of the innate immunity. The main component of this complex is called nucleotide oligomerization do- main–like receptor (NLR), an inter-cellular receptor for pathogenic and non-pathogenic signals. The other parts of complex consist of an adaptor protein apoptosis-as- sociated speck-like protein containing a caspase acti- vation and recruitment domain (ASC), and caspase-1 (Casp-1). The Inflammasome activation induces au- to-cleavage of pro-Caspase-1 into the it’s active form that leads to converting pro-Interleukin-1β (Pro-IL-1β) and pro-Interleukin-18 (Pro-IL-18) to the biological ac- tive forms (IL-1β and IL-18). These pro-inflammatory cytokines trigger other inflammatory cascades playing 1Department of Anatomy, School of medicine, Tehran University of Medical Sciences, Tehran, Iran. 2Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran. 3Department of Urology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran. 4Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran. 5Department of Anatomy, School of Medicine, Semnan University of Medical Sciences, Semnan, Iran. *Correspondence: Department of Anatomy, School of medicine, Tehran University of Medical Sciences, Poursina Avenue, 1417613151, Tehran, Iran. Tel: +98 (21) 88953008, Fax: +98 (21) 66419072, E-mail: abolhasf@sina.tums.ac.ir. Received July 2017 & Accepted September 2017 SEXUAL DYSFUNCTION AND ANDROLOGY Sexual Dysfunction and Infertility 5057 an important role in the innate immunity(10,11). Howev- er, higher activity of the inflammasome complex and resulted inflammation can induce damages in the in- volved tissues and cause a rapid pro-inflammatory form of cell death (Pyroptosis)(12). In 2011, Dulin et al. revealed disruption in Blood-Tes- tis-Barrier (BTB) and immune cell infiltration into the rat testis tissue following SCI. Accordingly, they found the elevation of IL-1β 72h post-injury(9). In a recent study, Fortune et al. assessed gene expression pat- tern and metabolomics in acute (24h) and chronic (3 months) phases of SCI in rat testis. They detected many transcripts and metabolites related to inflammatory, ox- idative stress and apoptotic pathways. Therefore, they concluded that an unstable niche have been established in testis after SCI causing SCI-dependent male infer- tility(13). Based on this background, SCI promotes an inflamma- tory microenvironment in the testis tissue leading to a large germ cells apoptosis. Inflammasome activation has not been reported in testis post-SCI in the literature. Therefore, we launched a time course study (Acute and chronic phases of SCI) regarding the role of essential genes (ASC and Casp-1) responsible for inflammasome complex in rat testis. MATERIALS AND METHODS Experimental animals In this study, 25 male Wistar rats (weight 200-250 g, age 8–10 weeks) were used in a random sampling de- sign. All animals were kept and maintained at 20–24 °C, 55 ± 10 % humidity and on a 12-hour light/dark cy- cle at the animal Laboratory Core Facility of Royan In- stitute, Tehran, Iran. They were fed by standard diet ad libitum, with access to tap water. All animal handlings, surgeries and cares were managed in compliance with the Tehran University of Medical Sciences ethics com- mittee. The animals acclimatized to the laboratory at least one week before surgery. Study design The rats were randomly divided into 5 groups and SCI induction and sacrifices were performed based on the study time-line (Figure 1). Contusion injury model at T10-T11 levels was chosen because there is no direct innervation from these levels to testes. Also, this model has the most similarity to traumatic injuries of the SCI patients in the clinics. Surgeries for contusion injury in- duction were done at four groups: SCI 1, SCI 3, SCI 7 and SCI 56. Rats in each group were killed and analyz- ed at a specific time point that is to say one day, three days, seven days and 56 days after surgery. One group remained intact as control (Co). SCI surgery The rats were anesthetized with an intraperitoneal ad- ministration of ketamine (80 mg/ kg) and xylazine (10 mg/kg) mixture. A dissection along the midline of the cord was performed on the 10th to 11th thoracic (T10– T11) vertebrae to create a 4mm longitudinal cut. After cutting the muscles and tissues, the laminectomy was performed. The vertebrae around T10 were stabilized and the contusion injury model was induced by NYU MASCIS (New York University Multicenter Animal Spinal Cord Injury Study) impactor (14,15). A 10-gram rod was dropped from a height of 25 mm. Complete contusion injury was obtained on spinal cord after 1-3 minutes (Figure 2). After surgery for recovery time, all rats were placed on a warm plate (38°C) for an hour. Manual bladder emptying was daily done from the day after surgery to remove residual urine, blood or any in- fection until the return of reflexive control of bladder function. Behavioral test From day one, all SCI rat models were functionally ex- amined. Open field locomotor test was performed by the non-invasive, Basso, Beattie, Bresnahan (BBB) lo- comotor rating scale(16). All animals with BBB scoring less than 2 were kept and others were removed from our study because of insufficient damage of spinal cord. Epididymis sampling The cauda epididymis was dissected before perfusion to avoid negative effects on sperm motility. This pro- cedure was done with care without any damage to ves- sels in that area. Epididymis, after one or two incision, minced in 1 mL pre-warmed Ham's F-10 medium (St. Louis, MO, USA) fortified with bovine serum albumin (BSA, Sigma, Louis, USA) for 30 to 45 min in a 37◦C incubator. Semen analysis After 30-45 min incubation, epididymal sperm released from the tissue and swim up into the medium. Sperm parameters were analyzed with a Computer-assisted Sperm Analyzer (CASA) as pointed out by Krause(17). The CASA system consisted of a phase contrast micro- scope (Eclipse E-200, Nikon Co., Japan) with a heat plate equipped with Sperm Class Analyzer® software Inflammasome gene expression in rat testes following SCI-Nikmehr et al. Table 1. Standard terminology for variables measured by computer-assisted sperm analyzer (CASA) systems. Parameters Unit Description Curvilinear velocity (VCL) μm/seconds Time-averaged velocity of a sperm head along its actual curvilinear path. Straight-line velocity (VSL) μm/seconds Time-averaged velocity of a sperm head along the straight line between its first detected position and its last Average path velocity (VAP) μm/seconds Time-averaged velocity of a sperm head along its average path Linearity (LIN) % The linearity of a curvilinear path, VSL/VCL Straightness (STR) % Linearity of the average path, VSL/VAP. Wobble (WOB) % A measure of oscillation of the actual path about the average path, VAP/VCL. Target gene Primer sequence (5′–3′) Annealing temperature(◦C) Gene bank code Product size(bp) Caspase-1 Forward CTTTCTGCTCTTCAACACCAG 59.61 NM_012762.2 122 Caspase-1 Reverse AATGTCCTCCAAGTCACAAGA 59.64 ASC Forward CCCATAGACCTCACTGATAAACT 59.55 NM_172322.1 127 ASC Reverse GCTCCAGACTCTTCCATAATCTT 60.13 Table 2. The sequences of rat specific primers for Caspase-1 and ASC cDNA. All primers were designed by Perlprimer v1.1.21. Vol 14 No 06 November-December 2017 5058 (SCA, full research version 5.1, Microptic Co., Bar- celona, Spain). In order to make sperm analysis, 4 μl sperm samples were placed in a standard count anal- ysis chamber (Leja, Nieuw Vennep Co., Netherlands). Specimens were observed with a Nikon microscope 10x/0.25 negative phase contrast field Ph1 BM, with an intermediate magnification of 0.7 and a green filter. At least 400 spermatozoa were counted for each sample. (Table1) Testis sampling and RNA extraction At each of the time points of study, the tissue reper- fusion was done with normal saline to eliminate blood from all tissues especially testes. After 45-60 min, tes- tes were dissected and cut into the three parts. Each part was snap frozen in liquid nitrogen and stored individu- ally in -80◦C, for further investigation. RNA extraction procedures were done under an RNase-free condition. Total RNA was isolated from testis samples using TRI- ZOL reagent (Sigma, St. Louis, MO, USA), based on the manufacturer’s protocol. Briefly, samples were warmed at lab temperature. Then, they were placed in 1.5 mL RNase free tubes and homogenized thoroughly with a needle. Afterward, 800 μl (1 ml per 50 to 100 mg tissue) TRIzol® Reagent was added to each tube and homogenized by hand with a tissue-homogenizer tip until tissue was completely dissociated. Then, 200 μl chloroform was added to each tube, capped tightly and shaken firmly. After 3 min incubation in lab tempera- ture, the tubes were centrifuged at 12000 rpm, 15min, and 4◦C. The aqueous (top) phase (containing RNA) was decanted in another RNase-free tube and the same volume of 100% isopropanol was added to the tube for RNA precipitation. Tubes are placed in -20◦C for an hour and centrifuged (12000 rpm, 15min, and 4◦C). The pellet was washed with 70% ethanol, air dried and dissolved in diethyl pyrocarbonate (DEPC) treated wa- ter. The extracted RNA was quantitated at 260 nm (Na- noDrop 2000 spectrophotometer, Thermo Scientific, Wilmington, DE). Quantitative Real-time PCR The isolated RNA was reversely transcripted to com- plementary DNA (cDNA) using Primescript RT re- agent kit (Takara Bio Inc., Otsu, Japan) according to the manufacturer guidelines. Primers were designed by PerlPrimer software version 1.1.21 (Marshall, 2004) and the sequences were listed in Table 2. The mRNA expression levels of the genes (ASC and Casp-1) were quantified using ABI/StepOnePlus Real-Time PCR System (Applied Biosystems). All real-time PCR assays were run in a total reaction volume of 20 μL. The result was shown as relative gene expression by the compara- tive Ct method ( 2─∆∆Ct )(18). All Ct values were deter- mined and normalized in comparison to a housekeeping gene (b-actin). Relative quantification was calculated by StepOneTM Real-Time PCR Software version 2.2 (Thermo Fisher Scientific, Waltham, MA). Statistical analysis All data were analyzed using SPSS statistical software version 22.0 (SPSS Inc., Chicago, IL). Results were ex- pressed as mean ± standard error of the mean (S.E.M). Analyses of the parametric data were done by one-way analysis of variance (ANOVA) with Turkey’s post hoc statistical tests. Non-parametric data were analyzed statistically using Kruskal-Wallis Test (nonparametric ANOVA) and Dunn's Multiple Comparisons for post- test. In all analyses, P < .05 was set as a significant lev- el. RESULTS Semen analysis Sperm parameters were evaluated by recruiting CASA system and statistically analyzed compared to those of the control group and P < 0.05 was considered statis- tically significant (Table 3). Sperm concentration was significantly lower in comparison to the control group, on day 1, 3 and 7 after SCI (P = .034, P = .002 and P Figure 1. The time line of the study. There were four groups that have undergone SCI surgery. Rats in each group were killed at a specific time point (Day1, Day3, Day7 and Day56) post injury and epididymis (for sperm analysis) and testis (for real-time PCR) were dissected. Groups Co SCI 1 SCI 3 SCI 7 SCI 56 Count (106 /ml) 27.64 ± 2.33 15.38 ± 1.41* 9.92 ± 0.77* 14.99 ± 1.47* 24.13 ± 4.95 Total Motility (%) 83.22 ± 3.52 52.09 ± 9.39 44.28 ± 4.58* 57.66 ± 0.93 52.19 ± 15.67 Progressive Motility (%) 64.11 ± 7.68 31.66 ± 7.44 19.11 ± 2.89* 28.02 ± 1.36* 42.24 ± 14.26 Non-progressive Motility (%) 19.10 ± 4.59 20.42 ± 4.82 25.16 ± 1.99 29.62 ± 0.70 9.72 ± 1.66 Immotile sperms (%) 16.79 ± 3.52 47.91 ± 4.70 55.71 ± 4.58* 42.34 ± 0.93 47.78 ± 15.64 VCL (%) 107.40 ± 12.61 53.71 ± 6.57* 35.65 ± 9.07* 41.03 ± 9.85* 76.60 ± 20.20 VSL (%) 28.62 ± 1.61 13.02 ± 3.69* 5.87 ± 1.20* 8.56 ± 2.74* 15.78 ± 5.30 VAP (%) 47.41 ± 3.96 26.37 ± 4.84 14.80 ± 4.24 16.99 ± 4.76 37.53 ± 10.78 LIN (%) 27.76 ± 3.60 23.30 ± 3.79 17.89 ± 1.98 18.72 ± 3.09 18.95 ± 2.84 *P < 0.05 compared to Co Table 3. Effects of SCI on sperm parameters in rats, on day 1, 3, 7 and 56 after injury. (Mean ± Standard error) Inflammasome gene expression in rat testes following SCI-Nikmehr et al. Sexual Dysfunction and Infertility 5059 = .028, respectively). After 56 days post injury, sperm count had returned almost to amount of control group. Total motility (%) had a decline in all groups, but it was significant just on 3 day group, compared to con- trol group (P = .02). Sperm progressive motility was re- duced significantly on day 3 and 7 after SCI (P = .01 and P=.034, respectively). Non-progressive motility had no significant differences at any time points, compared to that of control. The most immotile sperm number was observed on day 3, although there was a non-significant growth in immotile sperm percent at four-time points. VCL and VSL (%) significantly reduced on day 1, 3 and 7, respectively. But the most reduction was seen on day 3. There was a decrease in VAP and LIN levels at every mentioned time points post-injury, but the chang- es were not significant. ASC and Casp-1 mRNA Gene expression ASC and Casp-1 mRNA expression in rat testes of the control group, without any intervention, was at a low basic level (Figure 3, Co). In one day group the amount of mRNA expression level for ASC increased, but it was not significant. The ASC had a non-significant expression peak again, 56 days after SCI (Figure 3). Casp-1 also had an increased expression only one day after SCI. The peak level of Casp-1 was on day 3 (P = .009). After that, the expression level became lower, even on 56 days post-SCI. The correlation of gene expression and sperm param- eters The correlations were assessed between sperm parame- ters and gene expression (Figure 4). Sperm count cor- related negatively to Casp-1 expression (r = -0.555, P = .01). Moreover, there is a significant negative correla- tion between sperm progressive motility and Caspase-1 expression (r = -0.524, P = .02) DISCUSSION Impaired fertility is a common feature of men with SCI that is attributed to erectile and ejaculation dys- functions. In most cases, these problems are solvable with some methods like PVS and EEJ. However, semen quality of these patients is often poor. The majority of the studies have emphasized that low sperm motility and viability, leukocytospermia, and high sperm DNA fragmentation are common among SCI men. Many re- ports indicate that sperm count remained unchanged following SCI(6,19). In the present study, we examined sperm parameters of SCI rats at the acute (1,3,7 days after injury) and the chronic (56 days after injury) phas- es. Interestingly, sperm count fell by half just after one Figure 2. Rat model of spinal cord injury (T10-T11): A. Exposed spinal cord following a dorsal laminectomy procedure. B. bruising of the spinal cord after contusion induction Figure 3. Effects of SCI on gene expression of ASC and Caspase-1 in rat testis, on day, 1, 3, 7 and 56 post injury (*P < .05 compared to Co). Inflammasome gene expression in rat testes following SCI-Nikmehr et al. Vol 14 No 06 November-December 2017 5060 day and a third after 3 days post-injury. This severe re- duction was significant in the acute phase (1,3 and 7 days) but after 56 days (chronic phase) it had rebounded almost to the control group level. Sperm motility had a sharp decline after injury, as well. Total motility de- creased in the acute (1,3 and 7 days after injury) and the chronic (56 days after injury) phases of SCI but it was statistically significant just on day 3. The most effect of SCI was on progressive motility with a significant reduction on day 3 and 7. SCI caused a large increase in the percent of immotile sperm, especially on day 3 with a threefold increase. Most studies have analyzed sperm parameters post-SCI in human samples, and few researches have been con- ducted on the experimental models. In this field, the majority of studies indicated that sperm count after SCI was normal and most changes happen in sperm motility and viability(19). Interestingly, our study showed a rapid decline in sperm count in the acute phase of SCI. Low sperm motility in our research is in concordance with previous studies that they have been mentioned it as one of the most important causes of impaired fertility following SCI(19,20). However, some investigations have claimed seminal plasma of SCI men is toxic to sperm, and cauda epididymis and vas deferens(21,22) have sperm with better quality(23,24). In the present study, we analyz- ed sperm from caudal part of epididymis, to diminish toxic effects of ejaculated semen on sperm motility af- ter SCI. intriguingly, total motility dropped rapidly just after one day, and it remained low after 56 days from surgery. It seems that the greatest effect of SCI is on progressive motility, especially on day 3 and 7 post-in- jury (Acute phase). Although many reasons have been raised for male sub- fertility following SCI, spermatogenesis defects, large germ cell apoptosis and inflammatory conditions in tes- ticular tissue are not well-defined. In this regard, only a few investigations have been performed on experimen- tal models. Huang et al. carried out a time course study (3, 7, and 14 days after the SCI induction) on spermat- ogenesis abnormalities following SCI on male rats. They showed delayed spermiation and vacuolization of the nucleus of spermatids just 3 days after SCI. Other spermatogenic abnormalities were observed on day 14 group. Also, they demonstrated that hormone alteration is not the only reason for the impaired spermatogene- sis following SCI(25). Choobineh et al. reported exoge- nous testosterone therapy after SCI in adult mice could not compensate sexual hormone insufficiency and it caused reduction in natural testosterone production of testes(26). For the first time, Dulin et al. (2011) illustrat- ed that Blood-testis barrier (BTB) integrity was dis- rupted after SCI on male Sprague-Dawley rats. In that study, BTB became permeable to immunoglobulin G at both 72 hours and 10 months post SCI. The results indicated immune cell infiltration into the testis tissue and high expression of the pro-inflammatory cytokine IL-1β. Moreover, widespread germ cell apoptosis was observed at 72 h after SCI(9). In 2016, Fortune et al. showed many pathological events in testis in both acute and chronic phases of SCI. They revealed a pro-inflam- matory environment established after SCI in rat testis. Afterwards, other inflammatory cascades are activated resulting cell cycle dysregulation and apoptosis within the seminiferous tubules(13). Inflammasome is an inflammatory complex that is acti- vated under pathogenic and non-pathogenic conditions. Association of this complex with many diseases was previously detected(23-25), but it is not clearly defined in male infertility. Ibrahim et al. (2013) showed higher concentrations of some inflammasome indicators in- cluding IL-1◦, IL-18, Casp-1 and ASC in the semen of SCI affected men with chronic injury and even the in- flammasome suppression treatments have been applied to improve the semen quality in SCI patients(27,28). In this study, we evaluated the expression of ASC and Casp-1 in rat testis at the acute (1,3 and 7 days) and the chronic (56 days) phases of SCI. Interestingly, both of two genes were expressed higher than the control group, just after one day post-injury. ASC expression was high on day 1, dropped on day 3 and 7 and again raised on day 56 after injury but it was not significant at of the four time points of study. ASC is an adaptor pro- tein that makes a connection between NLRs and Casp- 1. However, recent investigations showed that some NLRs can be attached to Casp-1 directly without ASC (29). It means that ASC overexpression can indicate in- flammasome activation but is not necessary. Casp-1 is the best-known type of the inflammatory caspases and it is an indispensable component of in- flammasome complex. There are some pathways to activate the inflammasome complex (canonical and non-canonical), but in all pathways, Casp-1 is necessary for inflammasome formation and activation(30,31). In this study, Casp-1 expression increased more than two-fold on day 1 after injury and peaked on day 3 with a four- fold increase, compared to the control group. Following that, Casp-1 expression dropped on day 7 and remained Figure 4. The correlation of Casp-1 expression and sperm parameters of rat model of spinal cord injury. a. Casp-1 overexpression was significantly correlated to decline in sperm count (r = -0.555, P = .01). b. Sperm progressive motility reduced with increase in Casp-1 expression (r = -0.524, P = .02). Inflammasome gene expression in rat testes following SCI-Nikmehr et al. Sexual Dysfunction and Infertility 5061 unchanged on day 56 after injury. Since the Casp-1 is a direct marker of the inflammasome, it seems the SCI could activate inflammasome gene expression. Our data showed there are significant correlations be- tween some sperm parameters and inflammasome gene expression. Casp-1 expression negatively correlated with sperm count and progressive motility. The ex- pression of Casp-1 elevated after one day, and it was in the highest level on day 3 post-injury. As we know Casp-1 is the critical enzyme of the inflammasome complex (Interleukin-1◦/18 converting enzyme or ICE) activating pro-inflammatory cytokines of IL-1◦ and IL- 18. These cytokines have been known as the negative factors on the sperm parameters, especially on motility (22). In the current study, a sharp decline in sperm pro- gressive motility was detected on day 3 and 7 after inju- ry. It seems that this reduction could happen secondary to the Casp-1 overexpression on day 1 and 3. Identifi- cation of gene expression pattern of inflammasome in testis during the acute and the chronic phases of SCI is essential to therapeutic purposes. CONCLUSIONS ASC and Casp-1 are two inflammasome specific genes and are not related to other signaling pathways. There- fore, the expression of those genes on rat testis follow- ing SCI can indicate an abnormal cell situation. With these data, it seems there is a pattern in inflammasome gene expression in both acute and chronic phases of SCI. 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