Skeletal and Uterotrophic Effects of Endoxifen in Female Rats.

Endoxifen, the primary active metabolite of tamoxifen, is currently being investigated as a novel endocrine therapy for the treatment of breast cancer. Tamoxifen is a selective estrogen receptor modulator that elicits potent anti-breast cancer effects. However, long-term use of tamoxifen also induces bone loss in premenopausal women and is associated with an increased risk of endometrial cancer in postmenopausal women. For these reasons, we have used a rat model system to comprehensively characterize the impact of endoxifen on the skeleton and uterus. Our results demonstrate that endoxifen elicits beneficial effects on bone in ovary-intact rats and protects against bone loss following ovariectomy. Endoxifen is also shown to reduce bone turnover in both ovary-intact and ovariectomized rats at the cellular and biochemical levels. With regard to the uterus, endoxifen decreased uterine weight but maintained luminal epithelial cell height in ovariectomized animals. Within luminal epithelial cells, endoxifen resulted in differential effects on the expression levels of estrogen receptors α and ß as well as multiple other genes previously implicated in regulating epithelial cell proliferation and hypertrophy. These studies analyze the impact of extended endoxifen exposure on both bone and uterus using a Food and Drug Administration-recommended animal model. Although endoxifen is a more potent breast cancer agent than tamoxifen, the results of the present study demonstrate that endoxifen does not induce bone loss in ovary-intact rats and that it elicits partial agonistic effects on the uterus and skeleton in ovariectomized animals.

Chronic Phenotype Characterization of a Large-Animal Model of Hereditary Tyrosinemia Type 1.

Hereditary tyrosinemia type 1 (HT1) is an autosomal recessive disease caused by deficiency in fumarylacetoacetate hydrolase, the last enzyme in the tyrosine catabolic pathway. In this study, we investigated whether fumarylacetoacetate hydrolase deficient (FAH-/-) pigs, a novel large-animal model of HT1, develop fibrosis and cirrhosis characteristic of the human disease. FAH-/- pigs were treated with the protective drug 2-(2-nitro-4-trifluoromethylbenzoyl)-1, 3 cyclohexandione (NTBC) at a dose of 1 mg/kg per day initially after birth. After 30 days, they were assigned to one of three groups based on dosing of NTBC. Group 1 received ≥0.2 mg/kg per day, group 2 cycled on/off NTBC (0.05 mg/kg per day × 1 week/0 mg/kg per day × 3 weeks), and group 3 received no NTBC thereafter. Pigs were monitored for features of liver disease. Animals in group 1 continued to have weight gain and biochemical analyses comparable to wild-type pigs. Animals in group 2 had significant cessation of weight gain, abnormal biochemical test results, and various grades of fibrosis and cirrhosis. No evidence of hepatocellular carcinoma was detected. Group 3 animals declined rapidly, with acute liver failure. In conclusion, the FAH-/- pig is a large-animal model of HT1 with clinical characteristics that resemble the human phenotype. Under conditions of low-dose NTBC, FAH-/- pigs developed liver fibrosis and portal hypertension, and thus may serve as a large-animal model of chronic liver disease.

EUS-guided portal injection chemotherapy for treatment of hepatic metastases: feasibility in the acute porcine model.

BACKGROUND AND AIMS: Direct injection of chemotherapy into the portal vein for treatment of liver metastases may increase hepatic tissue levels while decreasing systemic levels and toxicities. We aimed to evaluate EUS-guided portal injection chemotherapy (EPIC) by using drug-eluting microbeads or nanoparticles and compare it with systemic injection. METHODS: We conducted a comparative feasibility trial in the acute porcine model (24 anesthetized pigs). Pigs were treated with irinotecan, doxorubicin, or albumin-bound paclitaxel nanoparticles (n = 8/group). Within each group, pigs were treated with EPIC or a systemic intravenous injection of drug and saline solution into the portal vein (n = 4/treatment). Irinotecan or doxorubicin were loaded onto microbeads for EPIC treatment only. We examined drug levels in tissue (1 hour) and plasma (15 minutes). RESULTS: EUS-guided access and injection was successful in all animals. EPIC with irinotecan-loaded microbeads showed nearly double the hepatic concentration compared with systemic injection (6242 vs 3692 ng/g) and almost half the systemic levels. EPIC with doxorubicin-loaded microbeads showed a 5-fold increase in hepatic levels (35,450 vs 6930 ng/g) and a 30-fold decrease in cardiac levels (153 vs 4805 ng/g) compared with systemic administration (P < .05 for both). EPIC with albumin-bound paclitaxel nanoparticles increased hepatic concentrations by 60% and decreased systemic levels by 24% to 32%. CONCLUSIONS: EPIC holds promise as a new treatment for hepatic metastases.

Low-Dose Gamma Irradiation of Decellularized Heart Valves Results in Tissue Injury In Vitro and In Vivo.

BACKGROUND: Decellularized heart valves are emerging as a potential alternative to current bioprostheses for valve replacement. Whereas techniques of decellularization have been thoroughly examined, terminal sterilization techniques have not received the same scrutiny. METHODS: This study evaluated low-dose gamma irradiation as a sterilization method for decellularized heart valves. Incubation of valves and transmission electron microscopy evaluation after different doses of gamma irradiation were used to determine the optimal dose of gamma irradiation. Quantitative evaluation of mechanical properties was done by tensile mechanical testing of isolated cusps. Sterilized decellularized heart valves were tested in a sheep model (n = 3 [1 at 1,500 Gy and 2 at 3,000 Gy]) of pulmonary valve replacement. RESULTS: Valves sterilized with gamma radiation between 1,000 Gy and 3,000 Gy were found to be optimal with in vitro testing. However, in vivo testing showed deteriorating valve function within 2 months. On explant, the valve with 1,500 Gy gamma irradiation showed signs of endocarditis with neutrophils on hematoxylin and eosin staining, and positive gram stain resembling streptococcus infection. The 3,000 Gy valves had no evidence of infection, but the hematoxylin and eosin staining showed evidence of wound remodeling with macrophages and fibroblasts. Tensile strength testing showed decreased strength (0 Gy: 2.53 ± 0.98 MPa, 1,500 Gy: 2.03 ± 1.23 MPa, and 3,000 Gy: 1.26 ± 0.90 MPa) with increasing levels of irradiation. CONCLUSIONS: Low-dose gamma irradiation does not maintain the mechanical integrity of valves, and the balance between sterilization and damage may not be able to be achieved with gamma irradiation. Other methods of terminal sterilization must be pursued and evaluated.

Pregnancy-associated plasma protein-A deficiency improves survival of mice on a high fat diet.

Obesity is on the rise in westernized countries, and visceral obesity in particular is associated with enhanced risk of developing metabolic disease and accelerated aging. Various dietary restriction regimens have been shown to extend healthy lifespan in a variety of species. However, identification of alternative approaches that could be more acceptable to humans is actively being pursued. We have shown previously that mice deficient in pregnancy-associated plasma protein-A (PAPP-A) have an extended healthy lifespan on a regular chow diet. In this study, we determined the lifespan of PAPP-A knock-out (KO) and wild-type (WT) littermates fed a high fat diet (HFD) starting at 12 months of age. PAPP-A KO and WT mice had equivalent weight gain as measured over 25 weeks on HFD. However, PAPP-A KO mice on HFD had a significant increase in lifespan (P=0.018). Body composition and tissue pathology were assessed in a separate cohort of mice after 30 weeks on HFD. Percent body fat was equivalent in the two groups. However, there was a decrease in visceral fat depot weights and an increase in serum adiponectin levels in PAPP-A KO compared to WT mice. Major pathological differences were seen in kidney, heart and testes, with PAPP-A KO mice having little, if any, evidence of inflammation, mineralization, or degeneration in these tissues compared to WT mice. Thus, PAPP-A is a novel drug target with the potential to promote healthy longevity without a need for dietary restriction.

Endoscopic band ligation for closure of GI perforations in a porcine animal model (with video).

BACKGROUND: GI perforations occur rarely during endoscopy but have life-threatening implications. OBJECTIVE: To evaluate endoscopic band ligation (EBL) for closure of acute GI perforations by using a porcine model. DESIGN: Investigator-initiated interventional pilot study by using an in vivo porcine model. SETTING: Tertiary-care institution. SUBJECTS: Ten domestic pigs. INTERVENTION: Each animal underwent a single endoscopic procedure, with creation of a single GI lumen perforation. Perforations of 10 to 20 mm were created in the esophagus, stomach, duodenum, and colon. EBL was used for closure. Fourteen days later, the pigs were killed, microbial cultures were obtained, and histologic review was done. MAIN OUTCOME MEASUREMENTS: Immediate and delayed endoscopic closure of the perforation site, evidence of clinical peritonitis during the 14-day follow-up. RESULTS: Ten pigs completed the protocol and survived without clinical peritonitis during the 14-day follow-up. Endoscopic closure of a 15-mm esophageal perforation failed, thus, no attempt was made to close a 20-mm esophageal perforation. Closure of all other perforations was successful. At necropsy, fibrinous peritonitis was suspected in one animal with a 10-mm duodenal perforation. Chronic inflammation and fibroplasia at the perforation sites were the most common histologic findings. LIMITATIONS: The applicability of widespread use in humans remains unknown despite successful case reports in the medical literature. CONCLUSION: EBL can be used successfully to close 10 to 20 mm perforations within normal stomach, duodenum, and colon and can prevent clinically relevant intra-abdominal infections. However, for esophageal perforations, closure may be limited to small (≤10 mm), iatrogenic perforations.

Mice deficient in PAPP-A show resistance to the development of diabetic nephropathy.

We investigated pregnancy-associated plasma protein-A (PAPP-A) in diabetic nephropathy. Normal human kidney showed specific staining for PAPP-A in glomeruli, and this staining was markedly increased in diabetic kidney. To assess the possible contribution of PAPP-A in the development of diabetic nephropathy, we induced diabetes with streptozotocin in 14-month-old WT and Papp-A knockout (KO) mice. Renal histopathology was evaluated after 4 months of stable hyperglycemia. Kidneys from diabetic WT mice showed multiple abnormalities including thickening of Bowman's capsule (100% of mice), increased glomerular size (80% of mice), tubule dilation (80% of mice), and mononuclear cell infiltration (90% of mice). Kidneys of age-matched non-diabetic WT mice had similar evidence of tubule dilation and mononuclear cell infiltration to those of diabetic WT mice, indicating that these changes were predominantly age-related. However, thickened Bowman's capsule and increased glomerular size appeared specific for the experimental diabetes. Kidneys from diabetic Papp-A KO mice had significantly reduced or no evidence of changes in Bowman's capsule thickening and glomerular size. There was also a shift to larger mesangial area and increased macrophage staining in diabetic WT mice compared with Papp-A KO mice. In summary, elevated PAPP-A expression in glomeruli is associated with diabetic nephropathy in humans and absence of PAPP-A is associated with resistance to the development of indicators of diabetic nephropathy in mice. These data suggest PAPP-A as a potential therapeutic target for diabetic nephropathy.

Downregulation of hematopoietic MUC1 during experimental colitis increases tumor-promoting myeloid-derived suppressor cells.

PURPOSE: MUC1 is a tumor-associated antigen that is aberrantly expressed in cancer and inflammatory bowel disease (IBD). Even though immune cells express low MUC1 levels, their modulations of MUC1 are important in tumor progression. Consistent with previous clinical data that show increased myeloid-derived suppressor cells (MDSCs) in IBD, we now show that downregulation of MUC1 on hematopoietic cells increases MDSCs in IBD, similar to our data in tumor-bearing mice. We hypothesize that MDSC expansion in IBD is critical for tumor progression. EXPERIMENTAL DESIGN: To mechanistically confirm the linkage between Muc1 downregulation and MDSC expansion, we generated chimeric mice that did not express Muc1 in the hematopoietic compartment (KO→WT). These mice were used in two models of colitis and colitis-associated cancer (CAC) and their responses were compared with wild-type (WT) chimeras (WT→WT). RESULTS: KO→WT mice show increased levels of MDSCs during colitis and increased protumorigenic signaling in the colon during CAC, resulting in larger colon tumors. RNA and protein analysis show increased upregulation of metalloproteinases, collagenases, defensins, complements, growth factors, cytokines, and chemokines in KO→WT mice as compared with WT→WT mice. Antibody-mediated depletion of MDSCs in mice during colitis reduced colon tumor formation during CAC. CONCLUSION: Development of CAC is a serious complication of colitis and our data highlight MDSCs as a targetable link between inflammation and cancer. In addition, the lack of MUC1 expression on MDSCs can be a novel marker for MDSCs, given that MDSCs are still not well characterized in human cancers.

Loss of the transcription factor GLI1 identifies a signaling network in the tumor microenvironment mediating KRAS oncogene-induced transformation.

Although the biological role of KRAS is clearly established in carcinogenesis, the molecular mechanisms underlying this phenomenon are not completely understood. In this study, we provide evidence of a novel signaling network regulated by the transcription factor GLI1 mediating KRAS-induced carcinogenesis. Using pancreatic cancer (a disease with high prevalence of KRAS mutations) as a model, we show that loss of GLI1 blocks the progression of KRAS-induced pancreatic preneoplastic lesions in mice with pancreas-specific Cre-activated oncogenic mutant kras. Mice lacking GLI1 develop only low-grade lesions at low frequency, and in most cases, the pancreata are histologically normal. Further characterization of the phenotype showed a decrease in the activation of STAT3 in pancreatic preneoplastic lesions; STAT3 is a transcription factor required for the development of premalignant lesions and their progression into pancreatic cancer. Analysis of the mechanisms revealed a key role for GLI1 in maintaining the levels of activated STAT3 through the modulation of IL-6 signaling. GLI1 binds to the IL-6 mouse promoter and regulates the activity and expression of this cytokine. This newly identified GLI1/IL-6 axis is active in fibroblasts, a known source of IL-6 in the tumor microenvironment. Sonic hedgehog induces GLI1 binding to the IL-6 promoter and increases IL-6 expression in fibroblasts in a paracrine manner. Finally, we demonstrate that mutant KRAS initiates this cascade by inducing the expression of Sonic hedgehog in cancer cells. Collectively, these results define a novel role for GLI1 in carcinogenesis acting as a downstream effector of oncogenic KRAS in the tumor microenvironment.

Detection of progressive myocardial tissue injury by ultrasonic integrated backscatter immediately after coronary reperfusion.

Myocardial reperfusion following ischemia may paradoxically cause additional injury, including microvascular damage and edema. These structural alterations augment tissue echogenicity, which is measurable by ultrasonic integrated backscatter (IB). We sought to characterize alterations in myocardial IB in an ischemic and reperfused region of the rat heart. Myocardial IB of the regions of interest in 12 adult male Sprague-Dawley rats was studied at baseline, during ischemia, and chronologically after coronary reopening, using an ultrasound frequency of 8 MHz. IB did not significantly change between baseline and ischemia. However, within 1 min of reperfusion, IB significantly increased and continued to increase until 10 min of reperfusion, when a plateau was reached. Areas of high echogenicity were comparable to infarcted areas on gross pathologic slices and had edema with extravasation of red blood cells. Myocardial reperfusion following ischemia significantly augments tissue echogenicity. A continuing increase of IB suggests a rapid progression of reperfusion injury.

Longevity and age-related pathology of mice deficient in pregnancy-associated plasma protein-A.

The pregnancy-associated plasma protein-A knockout (PAPP-A KO) mouse is a model of reduced local insulin-like growth factor (IGF)-I activity with normal circulating IGF-I levels. In this study, PAPP-A KO mice had significantly increased mean (27%), median (27%), and maximum (35%) life span compared with wild-type (WT) littermates. End-of-life pathology indicated that the incidence of neoplastic disease was not significantly different in the two groups of mice; however, it occurred in older aged PAPP-A KO compared with WT mice. Furthermore, PAPP-A KO mice were less likely to show degenerative changes of age. Scheduled pathologies at 78, 104, and 130 weeks of age indicated that WT mice, in general, had more degenerative changes and tumors earlier than PAPP-A KO mice. This was particularly true for abnormalities in heart, testes, brain, kidney, spleen, and thymus. In summary, the major contributors to the extended life span of PAPP-A KO mice are delayed occurrence of fatal neoplasias and decreased incidence of age-related degenerative changes.

Intravitreal alemtuzumab penetrates full-thickness retina in rabbit eyes.

PURPOSE: The purpose of this study was to assess whether alemtuzamab, a large antibody of 150 kDa, would be able to penetrate through the full-thickness retina of Dutch-belted rabbits. METHODS: Four Dutch-belted rabbits had intravitreal injections of alemtuzumab (1.5 mg in 0.05 mL). One rabbit each was killed at Day 1, Day 8, Day 15, and Day 29. The eyes were examined under frozen section and graded by immunostaining techniques for the degree of penetration of alemtuzumab into the retina. The degree of retinal staining was graded from 0 (no stain) to 4+ (marked stain). RESULTS: All study eyes showed antibody staining of the full-thickness retina as follows: 4+ at Day 1, 4+ at Day 8, 3+ at Day 15, and 2+ at Day 29. CONCLUSION: A 1.5-mg intravitreal dose of alemtuzumab was able to penetrate full-thickness retina throughout the full 29-day course of the study. Retinal toxicity studies are required before clinical use.

Reprogrammed FoxP3+ T regulatory cells become IL-17+ antigen-specific autoimmune effectors in vitro and in vivo.

Lymphocyte differentiation from naive CD4(+) T cells into mature Th1, Th2, Th17, or T regulatory cell (Treg) phenotypes has been considered end stage in character. In this study, we demonstrate that dendritic cells (DCs) activated with a novel immune modulator B7-DC XAb (DC(XAb)) can reprogram Tregs into T effector cells. Down-regulation of FoxP3 expression after either in vitro or in vivo Treg-DC(XAb) interaction is Ag-specific, IL-6-dependent, and results in the functional reprogramming of the mature T cell phenotype. The reprogrammed Tregs cease to express IL-10 and TGFbeta, fail to suppress T cell responses, and gain the ability to produce IFN-gamma, IL-17, and TNF-alpha. The ability of IL-6(+) DC(XAb) and the inability of IL-6(-/-) DC(XAb) vaccines to protect animals from lethal melanoma suggest that exogenously modulated DC can reprogram host Tregs. In support of this hypothesis and as a test for Ag specificity, transfer of DC(XAb) into RIP-OVA mice causes a break in immune tolerance, inducing diabetes. Conversely, adoptive transfer of reprogrammed Tregs but not similarly treated CD25(-) T cells into naive RIP-OVA mice is also sufficient to cause autoimmune diabetes. Yet, treatment of normal mice with B7-DC XAb fails to elicit generalized autoimmunity. The finding that mature Tregs can be reprogrammed into competent effector cells provides new insights into the plasticity of T cell lineage, underscores the importance of DC-T cell interaction in balancing immunity with tolerance, points to Tregs as a reservoir of autoimmune effectors, and defines a new approach for breaking tolerance to self Ags as a strategy for cancer immunotherapy.

Spontaneous vulvar papillomas in a colony of mice used for pancreatic cancer research.

Mice in a colony used for pancreatic cancer research and maintained in a barrier animal facility presented with vulvar masses. A census and examination of all colony animals was conducted on 17 February 2006; line, gender, and mass location were recorded; a slide caliper was used to measure the width, length, and height of each mass; and the volume of each mass was calculated. Progeny female mice from crossbreeding of the B6.FVB-Tg(Ipf1-cre)1Tuv and B6;129-Kras2tm4Tyj (KRAS(G12D/+)) strains presented with external vulvar and periauricular papillomas. The papillomas were present in 41.2% of all female crossbred mice and ranged in size from 8 to 36 mm3. Age of mice and tumor size were not correlated. Compared with the B6.FVB-Tg(Ipf1-cre)1Tuv line, the crossbred female mice were more likely to have a vulvar mass, with an odds ratio of 29.3, 95% confidence interval (1.5, 563.9) and a positive predictive value of 42.9%. Diagnostic evaluation, including electron microscopy, light microscopy, serology, and bacteriology, did not reveal a viral or other infectious etiology. Therefore, we speculate that interaction between the genetic background of the mice and the introduced Kras oncogene may be responsible for these papillomas.

Absence of histologic retinal toxicity of intravitreal nanogold in a rabbit model.

PURPOSE: To evaluate the retinal toxicity of intravitreal nanogold, a novel antiangiogenic and long-term delivery agent. METHODS: One eye of each of 16 Dutch-belted rabbits was injected with intravitreal nanogold; the other eye served as a control. Eight rabbits received a dose of 67 micromol/0.1 mL of nanogold intravitreally into 1 eye; the other 8 rabbits received a dose of 670 micromol/0.1 mL of nanogold intravitreally into 1 eye. Eight rabbits were killed at 1 week, and eight were killed at 1 month; both eyes of each rabbit were enucleated. The eyes were fixed with 2% paraformaldehyde and sectioned for histologic examination. RESULTS: In all injected and control eyes, there was mild vacuolization in the inner plexiform and ganglion cell layers. The retina and retinal pigment epithelium were otherwise histologically normal. CONCLUSION: Intravitreal nanogold at concentrations of 67 micromol/0.1 mL and 670 micromol/0.1 mL showed no signs of retinal or optic nerve toxicity by light microscopy during histologic examination at 1 month.

The MUC1 Cytoplasmic Tail and Tandem Repeat Domains Contribute to Mammary Oncogenesis in FVB Mice.

BACKGROUND: Though the importance of the transmembrane mucin MUC1 in mammary oncogenesis has long been recognized, the relative contributions of the cytoplasmic tail and tandem repeat domains are poorly understood. METHODS: To address this, mouse models of mammary carcinogenesis were created expressing full-length, cytoplasmic tail-deleted, or tandem repeat-deleted MUC1 constructs. RESULTS: Overexpression of full-length MUC1 resulted in tumor formation in young mice (≤12 months); however, loss of either the cytoplasmic tail or the tandem repeat domain abrogated this oncogenic capacity. Aged mice in all strains developed late-onset mammary tumors similar to those previously described for the FVB background. CONCLUSIONS: This study is the first spontaneous cancer model to address the relative importance of the cytoplasmic tail and tandem repeat domains to MUC1-driven mammary oncogenesis, and suggests that both of these domains are essential for tumor formation.

Submucosal endoscopy with mucosal flap safety valve.

BACKGROUND: There is no reliable endoscopic method to selectively resect deeper layers of the gut wall or to access the peritoneal cavity and prevent peritoneal soiling. OBJECTIVES: To determine the technical feasibility and safety of submucosal endoscopy with mucosal flap (SEMF) in accessing the peritoneal cavity through a large full-thickness gastric-muscle-wall resection. DESIGN: Ex vivo feasibility exploration and survival animal study. SETTINGS: Ex vivo samples were obtained from fresh harvested organs. In vivo procedures were conducted with the pigs under standard general anesthesia. INTERVENTIONS: High-pressure carbon dioxide (CO(2)) injection and balloon dissection created a large submucosal working space for insertion of a cap-fitted endoscope. By using the EMR cap, a full-thickness resection of the muscularis propria was performed. This full-thickness defect was sealed with the overlying mucosal flap and the use of hemoclips or tissue anchors. RESULTS: By using the SEMF technique in the ex vivo experiment, the gastric wall was successfully traversed in each stomach after submucosal dissection and full-thickness resection of the musclaris. Similarly, by using the SEMF technique in the in vivo procedures, the peritoneal cavity was successfully accessed and the defect was completely sealed by using the mucosal flap. All animals survived 1 week after the procedure. Ulceration was noted in 3 pigs, and a small bowel injury was noted in 1 pig. Leak testing was negative in all stomachs. CONCLUSIONS: By using the SEMF technique, submucosal space endoscopy and deep-layer gastric-wall resection were successfully performed. Furthermore, the mucosa overlying the dissected submucosal space served as a safe flap valve, preventing peritoneal leakage.

Absence of histologic retinal toxicity of intravitreal bevacizumab in a rabbit model.

PURPOSE: To evaluate the retinal toxicity of intravitreal bevacizumab in an animal model. DESIGN: Animal study. METHODS: Bevacizumab was injected into the vitreous of one eye of each of eight Dutch-belted rabbits; the other eye served as a control. Four rabbits received a dose of 1.25 mg/0.05 ml of bevacizumab intravitreally into one eye, and the other four rabbits were injected with 2.5 mg/0.1 ml of bevacizumab intravitreally into one eye. At one month, the rabbits were killed and both eyes enucleated. The eyes were fixed with paraformaldehyde 2% and examined by light microscopy. RESULTS: In all injected and control eyes, there was mild vacuolization in the ganglion cell layer, and disruption of photoreceptor outer segments in both treated and control eyes, to the same degree, consistent with autolysis. The optic nerve, retina, and retinal pigment epithelium were otherwise normal by light microscopy with no evidence of toxicity. CONCLUSIONS: Intravitreal bevacizumab at doses of 1.25 mg and 2.5 mg showed no signs of retinal or optic nerve toxicity by light microscopy in this rabbit model.