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Effect of ketotifen fumarate on experimental autoimmune orchitis and torsion of the spermatic cord

The aim of this work was to study effects of ketotifen fumarate (KF) on prevention of tissue damage in testes of rats with experimental autoimmune orchitis (EAO) and on the contralateral testis in a model of prolonged testicular cord torsion (TCT). Rats with EAO or TCT were injected intraperitoneally once daily with KF or saline solution (vehicle group). Incidence and severity of testicular damage were evaluated by histopathology using an EAO score or a Johnsen score. Mast cells (MC) were identified by histochemistry and quantified. In EAO model, KF significantly reduced severity of histopathological testicular damage compared to rats in the vehicle group. KF also reduced the number of testicular MC compared to vehicle group. Similarly, in TCT model, multifocal damage of the contralateral testis was observed 30 days after testicular torsion characterized by sloughing of the germinal epithelium, seminiferous tubule atrophy, and interstitial edema. Focal signs of inflammation and fibrosis of seminiferous tubular walls were also observed. In contrast, sections of contralateral testis of rats injected with KF and killed 30 days after surgery showed normal histological features. A significant decrease in the number of MC was observed in rats treated with KF compared to untreated animals. In conclusion, we demonstrated that treatment with KF reduced testicular inflammatory process and MC infiltrates in both EAO and TCT models. The results suggest a promising treatment for infertile male patients with testicular pathologies associated with inflammation and germ cell loss.


Induction of TCT and histopathology
Under anesthesia, a scrototomy on the left side and a 720° unilateral spermatic cord torsion was performed on Sprague-Dawley rats in the experimental (E) group. The testis was fixed into the scrotum using 4-0 silk suture. The rats were killed 30 days after surgery. At the end of each time segment, the contralateral testis was removed, weighed, fixed in Bouin's solution, dehydrated, and embedded in paraffin or quickly frozen to obtain cryostat sections for histopathology or immunohistochemistry, respectively. Paraffin-embedded testis sections (6 μm) were stained with hematoxylin-eosin Y. Tubular damage was quantified by a modified Johnsen score, which analyzes the degree of damage to seminiferous tubule germ cells by a score from 1 to 10. 25 Complete spermatogenesis with many spermatozoa is considered 10 and animals with a score under 9 were considered to have testicular damage. The mean tubular score was obtained by evaluating 50 ST per testis per rat. Tubular diameter was calculated in transversal testis sections with the aid of an ocular micrometer (8×, Kpl, Zeiss, Oberkochen, Germany).

Experimental design
In EAO model, a group of immunized rats was injected intraperitoneally with 1 or 4 mg kg −1 of KF (Roma Pharmacy and Laboratory S.A., Buenos Aires, Argentina) or vehicle (saline solution) once daily. Treatment started 48 h before the first immunization and continued daily throughout the experiment (treatment 1). The treatment of another group of rats was started 7 days after the last immunization (treatment 2). In TCT model, the rats were injected intraperitoneally with KF (1 mg kg −1 ) or vehicle once a day. Treatment was started from the moment of the surgical procedure and continued daily throughout the experiment. The rationale for the doses of KF used in this experimental design was based on prior in vivo study using similar doses of KF and the maximum recommended dose. 27

Histochemistry
To identify MC, testis sections were stained with Alcian blue (Sigma-Aldrich). MC were counted using a 25× objective, and the ocular (12.5×; 473014-9901, Zeiss, Oberkochen, Germany) was fitted with a quadratic grid with a total area of 96 100 μm 2 .

Delayed-type hypersensitivity (DTH)
DTH was measured by a footpad swelling test performed at the end of each experiment. The rats were intradermically challenged in the left footpad with 2.10 mg of TH supernatant in 50 μl saline. This fraction was obtained by TH centrifugation (PR-2 International Equipment Co., Boston, MA, USA) (20 000 g at 4°C). The other footpad was injected with the same volume of saline. Footpad thickness was measured with a micrometer (Teclock Corporation, Naruta-cho Okaya-shi NaganoPref., Japan) 48 h after the challenge. The results of footpad swelling were expressed as mean of at least three measurements per rat (in mm), calculated as the difference between the thickness of the HT-injected footpad and that of the saline injected footpad.

Statistical analyses
Comparisons of groups were assessed by the nonparametric Mann-Whitney U test or one-way ANOVA Kruskal-Wallis test as indicated in each figure's legend. P ≤ 0.05 was considered statistically significant.

Administration of KF reduced severity of EAO and epididymitis
We evaluated the effects of KF administration on rats during EAO. Two experimental designs were used. In treatment 1, KF was administered before the first immunization; in treatment 2, KF was administered after the last immunization when the first signs of disease became apparent 28,29 and continued until euthanasia. Histopathology showed that 100% of rats in the vehicle group developed orchitis (Figure 1a and 1b). In contrast, a decrease in EAO incidence and severity was observed in rats injected with KF (4 mg kg −1 ) in treatment 1 (80%) (Figure 1a). Interestingly, we also found that KF significantly reduced severity of epididymis inflammation compared to the vehicle group (Figure 2). Rats in the vehicle group presented severe orchitis with large areas of aspermatogenic ST, in which only spermatogonia and Sertoli cells attached to the tubular wall and interstitial inflammatory cell infiltrates intermingled with Leydig cells were present (Figure 1c). In contrast, most rats (90%) treated with KF showed ST with scarce germ cell sloughing or normal seminiferous epithelium (Figure 1d). Body weight of KF (4 mg kg −1 )-treated rats did not differ from that of untreated normal rats, suggesting that KF treatment induced no apparent adverse effects (P = 0.20) (mean ± standard error of the mean [s.e.m.] of body weight: untreated normal rats [n = 17], 342.90 ± 8.14 g; treatment 1 [n = 10], 336.50 ± 10.90 g; and treatment 2 [n = 6], 341.60 ± 14.90 g). Moreover, all rats survived after KF treatment. A significant reduction of inflammation and damage of ST was observed in KF treatment 1. In KF treatment 2, histopathology was similar in the vehicle and KF groups.

KF did not alter cell mediated immunity in vivo
We next investigated the effects of KF on T cell mediated immunity by analyzing in vivo DTH response to spermatic antigens. Footpad swelling observed in rats with treatment 1 (Figure 3a) and 2 (Figure 3b) was similar to that in rats treated with KF compared to those of vehicle groups.

KF treatment reduced MC numbers
To evaluate the effect of KF on testicular MC, sections from rats treated or untreated with KF were processed by Alcian blue staining. Figure 4a-4d illustrates the presence of MC near tunica albuginea. Quantitative assessment of Alcian blue-positive cells revealed that administration of KF to rats undergoing orchitis reduced the number of MC compared to vehicle-treated rats (P = 0.020; Figure 4e).

Administration of KF prevented histological damage in the contralateral testis after prolonged TCT
Multifocal damage of the contralateral testis was observed 30 days after testicular torsion in the vehicle group. Testis damage was characterized by sloughing of the germinal epithelium, seminiferous tubule atrophy, and interstitial edema. Focal signs of inflammation and fibrosis of seminiferous tubular walls were also observed (Figure 5a). In contrast, contralateral testis sections of rats treated with KF for 30 days after surgical torsion showed normal histology (Figure 5b). Comparison of tubular diameter and Johnsen score showed significant differences between KF and vehicle group (P = 0.003 and P = 0.008, respectively; Figure 5c and 5d).

KF reduced the number of MC in the contralateral testis after prolonged TCT
MC were observed near the tunica albuginea and vessels (Figure 6a-6d). The number of MC decreased significantly in the Rats with EAO treated with saline (vehicle) or ketotifen fumarate were tested for DTH to testicular antigens during (a) treatment 1 and (b) treatment 2. Two days before euthanasia (day 54), TH was injected into rats' left footpad. The right footpad was injected with saline. Footpad swelling was measured after 48 h and results are expressed as the mean difference between the thickness of the HT-injected footpad and that of the saline-injected footpad ± standard error of the mean (s.e.m.) in each group (nonparametric one-way ANOVA Kruskal-Wallis test). EAO: experimental autoimmune orchitis; DTH: delayed-type hypersensitivity. b a Figure 1: Impact of ketotifen fumarate on incidence and severity of EAO score. All rats were immunized with testicular homogenate and adjuvants. A group of rats was injected with saline solution (vehicle) or ketotifen fumarate.

DISCUSSION
Inflammatory processes in the testis are frequently associated with infertility. 9 In fact, a high percentage of patients with subfertility or infertility present different degrees of testicular immune cell infiltrates. 30 TCT and EAO are interesting models to study testicular inflammation and infertility. Testicular inflammatory infiltrates are composed mainly of dendritic cells, macrophages, lymphocytes, and MC. 12,16,25 The inflammatory process and oxidative stress generate germ cell sloughing and apoptosis, resulting in aspermatogenesis and atrophy of ST. 19,31 MC have a key role in inflammation and tissue fibrosis in several models of autoimmune diseases. 32,33 In vitro, MC can promote fibroblast proliferation and collagen synthesis. 34 Presynthesized substances such as histamine, chymase, tryptase, carboxipeptidase A, and tumor necrosis factor-alpha (TNFα) are stored in granules and released immediately after MC activation. TNFα, interleukin (IL) 6, and IL1b are de novo synthesized after MC activation. 35 Because the present work focuses on MC, we evaluated the effect of KF, a high affinity blocker of histamine H1 receptor, on testicular inflammation and spermatogenesis in TCT and EAO experimental models.
Although a small number of MC are present in the normal mammal testis, in inflammatory conditions, the number of MC increases. [35][36][37] Testicular MC are also associated with sclerosis and fibrosis of ST in patients with varicocele and defective spermatogenesis. 38,39 In the TCT model, multifocal damage of the contralateral testis characterized by focal signs of inflammation and fibrosis of seminiferous tubular walls was observed 30 days after testicular torsion in the vehicle group. In contrast, the present results showed normal testicular histology in rats treated with 1 mg kg −1 day −1 of KF and a significantly decreased number of MC in the contralateral testis of rats treated with KF compared to the vehicle group. Similar to our findings, Acikgoz et al. 27 observed that the number of MC decreased in the contralateral descended testis in rats treated with MC blocker in experimental unilateral undescended testis, supporting its therapeutic use for autoimmune testicular inflammation.
In the EAO model, we also observed decreased incidence and severity of orchitis and epididymal inflammation in rats injected with 4 mg kg −1 day −1 of KF compared to the vehicle. We observed no adverse effects on weight or behavior in rats treated with KF in spite of the high doses used. By analyzing in vivo DTH response to spermatic antigens, we showed that KF treatment does not modify cell mediated immunity. In effect, this drug is mainly an immunomodulator with anti-histaminic action but does not block molecular steps that activate cell-mediated immunity.
Reduction of the number of MC is associated with reduction of testicular damage and improved spermatogenesis. The results suggest that KF inhibits testicular MC degranulation preventing the release of inflammatory and fibrotic mediators, consequently improving histopathological parameters. MC are a source of numerous cytokines, chemokines, and growth factors with potential autocrine, paracrine, local, and systemic effects. 40 In fact, some of these, such as TNFα, IL6, C-C chemokine ligand (CCL) 2, and CCL4, play a relevant role in the testis during inflammation. 17 KF, like other MC stabilizers, was reported to restrain serum TNFα levels and improve survival in a murine experimental model of sepsis. 41 KF also has a very important role in regulating macrophage metabolic activity, modulating nitric oxide (NO) production by inhibition of inducible oxide nitric synthase (iNOS) expression in lipopolysaccharide-stimulated cells. This inhibition is followed by a significant reduction in nitrite levels. This drug had no scavenger activity against NO in the chemical system. Therefore, it is possible that the inhibitory effect on macrophage iNOS expression could be due to its ability to affect intracellular signaling pathways leading NO production. 42 MC act in concert with macrophages and Leydig cells, germ cells, and peritubular cells which also express histamine receptor H1. 43,44 Histamine has also been reported to have an important role in controlling Leydig cell steroidogenesis. 44 Hence, we cannot exclude the possibility that KF as an antagonist of H1 may modulate other parameters beyond those we describe in this report.
MC blockers have been used empirically for the treatment of male infertility with controversial results. Yamamoto et al. 45 reported a significant improvement of semen parameters and pregnancies in a group of oligozoospermic patients treated with tranilast, and Matsuki et al. 46 reported similar results in infertile male patients treated with the MC blocker ebastine. However, Cayan et al. 47 reported that treatment with an antihistamine drug, fexofenadine, in infertile patients with a significant increase of testicular MC appears to have no benefit; no data concerning semen quality and pregnancies were reported. Use of this drug was discontinued due to lack of homogeneous results from different authors. Recently, Mondillo et al. 48 summarized the negative impact of anti-histamines loratadine, desloratadine, cimetidine, and ranitidine on male reproduction.
In contrast, several authors reported that KF treatment of infertile patients with idiopathic oligozoospermia and asthenozoospermia resulted in moderate, but statistically significant improvement in sperm count and motility and reduction of leukocytospermia and necrospermia. 3,5,49 Finally, in a preliminary study, we observed a significant improvement in seminal parameters and pregnancy rates after intracytoplasmic sperm injection (ICSI) in male patients with severe oligo-asthenozoospermia treated with KF. 50

CONCLUSION
Our results indicate that MC are involved in the pathogenesis of testicular inflammatory process. We demonstrated that treatment with KF can significantly reduce testis damage in experimental models of orchitis and prevent impairment of contralateral testis after testicular torsion. The present work enlarges the use of KF as treatment in testicular pathologies associated with inflammation and germ cell loss.

AUTHOR CONTRIBUTIONS
DM performed EAO experiments and was involved in interpretation of results. CMS contributed with histochemistry experiments. LL was involved in the study's conception, interpretation and discussion of data, and contributed to drafting of the article. MGRP performed TCT experiments and contributed to drafting of the article. VAG performed experiments and was involved in the study's conception and design, interpretation and discussion of data, and drafting of the article. All authors read and approved the final manuscript.