A protocol for creating endometriosis in rhesus macaques (Macaca mulatta).

BACKGROUND: Endometriosis is the presence of endometrium-like tissue outside the uterine cavity. An experimental model of endometriosis has been created in the baboon by the transcervical collection and laparoscopic inoculation of menstrual endometrium. Macaques are the preferred model for pharmaceutical development, but the complex anatomy of the macaque cervix makes the baboon method impractical. In this work, we sought to validate a surgical approach for creating endometriosis in macaques. METHODS: Menstrual endometrium was collected via laparoscopic intrauterine puncture and transferred to the peritoneal cavity. We repeated this procedure during three menstruations. Endometriotic tissue was identified during laparoscopy, collected, and characterized by immunohistochemistry. RESULTS: Sham surgery-treated animals (n = 3) failed to develop endometriosis. We identified red, powder burnt, and white lesions in 13/14 of the treated animals; the stroma of the red lesions stained positive for ovarian steroid receptors. CONCLUSION: This surgical technique can reliably create hormone-responsive endometriosis in macaques for therapeutic studies.

Vasocutaneous fistula formation and repair following inguinal hernia repair in a rhesus monkey (Macaca mulatta).

A 6-year-old adult male rhesus macaque (Macaca mulatta) developed a vasocutaneous fistula following an anatomic inguinal hernia repair years earlier. The vasocutaneous fistula was surgically repaired, the vas deferens was ligated, and the wound was closed in layers with non-overlapping suture lines with no further adverse sequalae of events.

Successful implementation of intestinal resection and anastomosis in non-human primates suggests the possibility of longitudinal intestinal research.

Intestinal biopsy is a basic experimental method for studying pathological changes in the intestinal tract during human immunodeficiency virus (HIV) infection. In this study, jejunal resection and anastomosis were successfully performed in 12 Chinese rhesus macaques ( Macaca mulatta). The sampled gut tissues were then examined by hematoxylin and eosin (H&E) staining, electron microscopy, flow cytometry, immunofluorescence detection, and RNA quality analysis to ensure suitability for histological, physiological, pathological, and immunological detection, as well as mechanistic analysis at the cellular and molecular level. Importantly, the surgery did not affect the ratio or number of immune cells in peripheral blood or the concentration of lipids, proteins, and vitamins in plasma, which are important indicators of nutritional status. Our results thus indicated that jejunal resection and anastomosis are feasible, and that immune homeostasis and intestinal barrier integrity are not altered by surgery. All macaques recovered well (except for one), with no postoperative complications. Therefore, this animal surgery may be applicable for longitudinal intestinal research related to diseases such as acquired immunodeficiency syndrome (AIDS).

Bioinformatic analysis of peripheral blood RNA-sequencing sensitively detects the cause of late graft loss following overt hyperglycemia in pig-to-nonhuman primate islet xenotransplantation.

Clinical islet transplantation has recently been a promising treatment option for intractable type 1 diabetes patients. Although early graft loss has been well studied and controlled, the mechanisms of late graft loss largely remains obscure. Since long-term islet graft survival had not been achieved in islet xenotransplantation, it has been impossible to explore the mechanism of late islet graft loss. Fortunately, recent advances where consistent long-term survival (≥6 months) of adult porcine islet grafts was achieved in five independent, diabetic nonhuman primates (NHPs) enabled us to investigate on the late graft loss. Regardless of the conventional immune monitoring methods applied in the post-transplant period, the initiation of late graft loss could rarely be detected before the overt graft loss observed via uncontrolled blood glucose level. Thus, we retrospectively analyzed the gene expression profiles in 2 rhesus monkey recipients using peripheral blood RNA-sequencing (RNA-seq) data to find out the potential cause(s) of late graft loss. Bioinformatic analyses showed that highly relevant immunological pathways were activated in the animal which experienced late graft failure. Further connectivity analyses revealed that the activation of T cell signaling pathways was the most prominent, suggesting that T cell-mediated graft rejection could be the cause of the late-phase islet loss. Indeed, the porcine islets in the biopsied monkey liver samples were heavily infiltrated with CD3+ T cells. Furthermore, hypothesis test using a computational experiment reinforced our conclusion. Taken together, we suggest that bioinformatics analyses with peripheral blood RNA-seq could unveil the cause of insidious late islet graft loss.

Virtual Rhesus Labyrinth Model Predicts Responses to Electrical Stimulation Delivered by a Vestibular Prosthesis.

To better understand the spread of prosthetic current in the inner ear and to facilitate design of electrode arrays and stimulation protocols for a vestibular implant system intended to restore sensation after loss of vestibular hair cell function, we created a model of the primate labyrinth. Because the geometry of the implanted ear is complex, accurately modeling effects of prosthetic stimuli on vestibular afferent activity required a detailed representation of labyrinthine anatomy. Model geometry was therefore generated from three-dimensional (3D) reconstructions of a normal rhesus temporal bone imaged using micro-MRI and micro-CT. For systematically varied combinations of active and return electrode location, the extracellular potential field during a biphasic current pulse was computed using finite element methods. Potential field values served as inputs to stochastic, nonlinear dynamic models for each of 2415 vestibular afferent axons, each with unique origin on the neuroepithelium and spiking dynamics based on a modified Smith and Goldberg model. We tested the model by comparing predicted and actual 3D vestibulo-ocular reflex (VOR) responses for eye rotation elicited by prosthetic stimuli. The model was individualized for each implanted animal by placing model electrodes in the standard labyrinth geometry based on CT localization of actual implanted electrodes. Eye rotation 3D axes were predicted from relative proportions of model axons excited within each of the three ampullary nerves, and predictions were compared to archival eye movement response data measured in three alert rhesus monkeys using 3D scleral coil oculography. Multiple empirically observed features emerged as properties of the model, including effects of changing active and return electrode position. The model predicts improved prosthesis performance when the reference electrode is in the labyrinth's common crus (CC) rather than outside the temporal bone, especially if the reference electrode is inserted nearly to the junction of the CC with the vestibule. Extension of the model to human anatomy should facilitate optimal design of electrode arrays for clinical application.

Single-photon Emission Computed Tomography-Computed Tomography Using 99mTc-labeled Leukocytes for Evaluating Infection Associated with a Cranial Implant in a Rhesus Macaque (Macaca mulatta).

An adult male rhesus macaque (Macaca mulatta) that was enrolled in a study evaluating cognition and memory presented with suppurative exudate along the margins of a long-standing cranial implant that included a stainless-steel head post, plastic left-sided recording cylinder, and acrylic over a previously placed right-sided recording cylinder. Both cylinders were located at the level of the prefrontal cortex. After treatment comprising systemic antibiotics and daily cleaning with povidone-iodine for several months, the macaque underwent single-photon emission computed tomography-computed tomography (SPECT-CT) in which his neutrophils were labeled with 99mTc-hexamethylpropylene amine oxime ( 99m Tc-HMPAO) to evalu- ate for active infection below the implant. Soft tissue inflammation and osteomyelitis were found at the site of the previous right-sided recording cylinder. Cephalosporin and tetracycline antibiotics were administered for 12 wk. Follow-up SPECT-CT imaging was then performed to evaluate response to medical treatment. Results indicated no change in the degrees of soft tissue inflammation and osteomyelitis associated with the right-sided recording cylinder site. SPECT-CT imaging was used to guide the surgical removal of the implant and debridement of the infected tissue. On removal of the entire cranial implant, the osteomyelitis and soft tissue inflammation observed on the pre- and posttreatment SPECT-CT scans were confirmed. In addition, a large cavitary defect through the calvarium with suppurative exudate was discovered below the base of the head post. Infection in this defect was not apparent on SPECT; however, the bony defect was confirmed on reevaluation of the CT images. We concluded that the infection in this defect was silent on SPECT due to the limited vascularization of the sur-rounding bone and the chronicity of the infection. This case study is the first to describe the use of SPECT-CT for evaluating soft tissue inflammation and osteomyelitis beneath a cranial implant in a NHP.

Assessment of pre-operative maropitant citrate use in macaque (Macaca fasicularis & Macaca mulatta) neurosurgical procedures.

BACKGROUND: Retrospective analysis of post-operative vomiting (POV) in non-human primates at our institution was 11%. Based on this additional risk factor for post-operative complications, we aimed to eliminate or decrease POV by adding an antiemetic, maropitant citrate, to the pre-medication protocol. METHODS: Retrospective and prospective data were collected over a 5-year period from 46 macaques of two species during 155 procedures. Additionally, blood was collected from five Macaca mulatta to perform a pharmacokinetic analysis. RESULTS: A 1 mg/kg subcutaneous dose of maropitant given pre-operatively significantly decreased POV. Findings indicated post-neurosurgical emesis in Macaca fasicularis was significantly greater than in Macaca mulatta. Pharmacokinetic analysis of maropitant in Macaca mulatta determined the mean maximum plasma concentration to be 113 ng/mL. CONCLUSIONS: Maropitant administration prior to anesthesia for neurosurgeries decreased our incidence of POV to 1%. The plasma concentration reaches the proposed plasma level for clinical efficacy approximately 20 minutes after administration.

The use of desflurane for neurosurgical procedures in rhesus macaque ( Macaca mulatta).

Volatile agents are widely used to anaesthetise laboratory non-human primates as they allow a rapid induction and recovery as well as an easy adjustment of the anaesthesia plan. Desflurane is currently the volatile agent with the lowest solubility in blood, and hence enables the most rapid onset of anaesthesia and most rapid recovery. This study aimed to investigate the suitability of desflurane for maintenance of general anaesthesia in rhesus macaques undergoing elective experimental neurosurgery. Fourteen primates (five males and nine females) were sedated with ketamine (10 mg kg-1) and anaesthesia was induced with propofol (usually 8 mg kg-1 IV). Anaesthesia was maintained with desflurane (5.9 ± 0.8 %) and alfentanil (0.2-0.5 µg kg-1 min-1 IV). Animals were mechanically ventilated. Meloxicam (0.3 mg kg-1) and methylprednisolone infusion (5.4 mg kg-1 h-1) were also administered. All the primates were successfully anaesthetised and no severe complications related to the procedure or the anaesthesia regimen occurred. No major differences in physiological parameters and recovery times between the male and female groups were found. Emergence from anaesthesia was rapid (male 5.2 ± 2.4 min; female 4.1 ± 1.7 min) but its quality was assessed as equivalent to two other volatile anaesthetics, isoflurane and sevoflurane. These had previously been assessed for neuroanaesthesia in rhesus macaques. In conclusion, this study demonstrated that desflurane was suitable for maintenance of general anaesthesia for elective experimental neurosurgical procedures in rhesus macaque. However the vasodilatory action of the desflurane may limit its use in cases of severe intracranial hypertension or systemic hypotension.

Recent refinements to cranial implants for rhesus macaques (Macaca mulatta).

The advent of cranial implants revolutionized primate neurophysiological research because they allow researchers to stably record neural activity from monkeys during active behavior. Cranial implants have improved over the years since their introduction, but chronic implants still increase the risk for medical complications including bacterial contamination and resultant infection, chronic inflammation, bone and tissue loss and complications related to the use of dental acrylic. These complications can lead to implant failure and early termination of study protocols. In an effort to reduce complications, we describe several refinements that have helped us improve cranial implants and the wellbeing of implanted primates.

The mandibles of castrated male rhesus macaques (Macaca mulatta): The effects of orchidectomy on bone and teeth.

OBJECTIVES: The objectives of this study were to evaluate the long-term effects of orchidectomy and low testosterone on the craniofaciodental development and maintenance of skeletal and oral health in rhesus macaques. MATERIALS AND METHODS: Mandibles of four castrated and intact age-matched male rhesus monkeys (Macaca mulatta) from Cayo Santiago were compared for mandibular morphology and teeth, abnormalities, pathology, and cortical bone thickness and density using a digital sliding caliper and analysis of three-dimensional X-ray images. RESULTS: Although all four castrates were generally comparable to intact males in overall mandible and teeth size, there were some significant differences. In the castrates, (1) the distance between the two rami was narrower than in intact males leading to a relatively narrower and longer face; (2) both the mandibular body and ramus had thinner cortical bone leading to less total bone mass; and (3) the canines and molar teeth were slender with lower and less robust tooth cusps. In addition, the alveolar bone of two geriatric castrates was greatly receded with signs of periodontitis more severe than in intact aged males. Old castrates also had severe temporomandibular joint osteoarthritis. DISCUSSION: The findings suggest the importance of testosterone in craniofaciodental development, and maintenance of skeletal and oral health in male macaques. These results suggest that dental health professionals might want to include in their medical history questionnaires whether or not male patients have taken hormone (testosterone) replacement therapy.

Effect of long-term castration on serum biochemistry in rhesus monkeys.

BACKGROUND: Testicular failure has an effect on normal physiology. To address this issue, an experimental non-human primate model of long-term castrated rhesus monkey was chosen for this study to evaluate the influence of castration on various biochemical parameters. METHODS: Nine castrated rhesus monkeys were evaluated for changes in body weight, serum testosterone, and serum biochemical parameters as compared to those in non-castrated macaques. RESULTS: Castration caused statistically significant changes in body weight, biochemical analytes, and testosterone levels. Body weight and testosterone levels were decreased, and there were increase in alanine aminotransferase, cholesterol, serum bilirubin, phosphorous, alkaline phosphatase, urea and a decrease in serum protein, uric acid, creatinine, and triglycerides. CONCLUSIONS: This study provided essential baseline information on biochemical variables due to the effect of castration associated with declining levels of testosterone, as data are not readily accessible from the existing body of scientific literature on non-human primates.

Percutaneous transhepatic portal catheterization guided by ultrasound technology for islet transplantation in rhesus monkey.

BACKGROUND: Pig islet xenotransplantation has the potential to overcome the shortage of donated human islets for islet cell transplantation in type 1 diabetes. Testing in non-human primate models is necessary before clinical application in humans. Intraportal islet transplantation in monkeys is usually performed by surgical infusion during laparotomy or laparoscopy. In this paper, we describe a new method of percutaneous transhepatic portal catheterization (PTPC) as an alternative to current methods of islet transplantation in rhesus monkeys. METHODS: We performed ultrasound-guided PTPC in five adult rhesus monkeys weighing 7-8 kg, with portal vein catheterization confirmed by digital subtraction angiography. We monitored for complications in the thoracic and abdominal cavity. To evaluate the safety of ultrasound-guided PTPC, we recorded the changes in portal pressure throughout the microbead transplantation procedure. RESULTS: Ultrasound-guided PTPC and infusion of 16 000 microbeads/kg body weight into the portal vein was successful in all five monkeys. Differences in the hepatobiliary anatomy of rhesus monkeys compared to humans led to a higher initial complication rate. The first monkey died of abdominal hemorrhage 10 hours post-transplantation. The second suffered from a mild pneumothorax but recovered fully after taking only conservative measures. After gaining experience with the first two monkeys, we decreased both the hepatic puncture time and the number of puncture attempts required, with the remaining three monkeys experiencing no complications. Portal pressures initially increased proportional to the number of transplanted microbeads but returned to pre-infusion levels at 30 minutes post-transplantation. The changes in portal pressures occurring during the procedure were not significantly different. CONCLUSIONS: Ultrasound-guided PTPC is an effective, convenient, and minimally invasive method suitable for use in non-human primate models of islet cell transplantation provided that care is taken with hepatic puncture. Its advantages must be weighed against the risks of procedure-related complications.

Pancreas anatomy and surgical procedure for pancreatectomy in rhesus monkeys.

BACKGROUND: The aim of this study was to investigate the pancreas anatomy and surgical procedure for harvesting pancreas for islet isolation while performing pancreatectomy to induce diabetes in rhesus monkeys. METHODS: The necropsy was performed in three cadaveric monkeys. Two monkeys underwent the total pancreatectomy and four underwent partial pancreatectomy (70-75%). RESULTS: The greater omentum without ligament to transverse colon, the cystic artery arising from the proper hepatic artery and the branches supplying the paries posterior gastricus from the splenic artery were observed. For pancreatectomy, resected pancreas can be used for islet isolation. Diabetes was not induced in the monkeys undergoing partial pancreatectomy (70-75%). CONCLUSIONS: Pancreas anatomy in rhesus monkeys is not the same as in human. Diabetes can be induced in rhesus monkeys by total but not partial pancreatectomy (70-75%). Resected pancreas can be used for islet isolation while performing pancreatectomy to induce diabetes.

Transurethral bladder catheterization of male rhesus macaques: a refinement of approach.

BACKGROUND: Successful transurethral bladder catheterization in male non-human primates can be challenging. An optimized approach for consistent and reproducible catheterization using a refined technique is described. METHODS: Under sedated and non-sedated conditions, transurethral bladder catheterization was performed on 25 male rhesus macaques of varying ages and body weights over time. A refined technique ensuring optimal lubrication of the urethral canal prior to catheter insertion was utilized along with various single and multiple lumen catheters. RESULTS: All animals were successfully catheterized. Sixty-five catheterization sessions were conducted with a high overall success rate (100%). The incidence of catheter (10%) and post-catheterization (2%) complications was low. CONCLUSIONS: The urinary bladder of male rhesus can be reliably and reproducibly catheterized with minimal complication using this approach. Successful catheterization was facilitated by thorough urethral lubrication and using suitable catheters. In addition, this approach may be performed without sedation on thoroughly conditioned animals.

Refinement of vascular access port placement in nonhuman primates: complication rates and outcomes.

Chronic vascular access is often needed in experimental animal studies, and vascular access ports (VAP) have been proposed as an alternative to conventional venipuncture. We previously reported on VAP implantation by using femoral venous cutdown (FVC) and tunneling. In an attempt to decrease the moderate complications associated with the FVC method, we developed the single-incision, peripheral-insertion (SIPI) method. In a retrospective evaluation, 92 FVC procedures were compared with 113 SIPI procedures in cynomolgus and rhesus macaques and baboons with as much as 2.5 y of follow-up. The rate of complications was significantly lower for the SIPI method than for the FVC method (19.4% versus 33.7%), particularly in regard to infectious complications (8.0% versus 27.3%, respectively). In addition, VAP patency for blood sampling and fluid infusion was significantly better for the SIPI method than for the FVC method, with 1-y patency rate of 83% and 46%, respectively, and 2-y patency rate of 74% and 36%, respectively. Additional advantages of the SIPI method include the simplified implantation of the VAP and access in the homecage without any sedation or restraint after appropriate training of animals to cooperate. We conclude that the SIPI method presents an opportunity for refinement and is superior to the FVC method for chronic vascular access.

A new model of cortical stroke in the rhesus macaque.

Primate models are essential tools for translational research in stroke but are reportedly inconsistent in their ability to produce cortical infarcts of reproducible size. Here, we report a new stroke model using a transorbital, reversible, two-vessel occlusion approach in male rhesus macaques that produces consistent and reproducible cortical infarcts. The right middle cerebral artery (distal to the orbitofrontal branch) and both anterior cerebral arteries were occluded with vascular clips. Bilateral occlusion of the anterior cerebral artery was critical for reducing collateral flow to the ipsilateral cortex. Reversible ischemia was induced for 45, 60, or 90 mins (n=2/timepoint) and infarct volume and neurologic outcome were evaluated. The infarcts were located predominantly in the cortex and increased in size with extended duration of ischemia determined by T(2)-weighted magnetic resonance imaging . Infarct volume measured by 2,3,5-triphenyl tetrazolium chloride and cresyl violet staining corroborated magnetic resonance imaging results. Neurologic deficit scores worsened gradually with longer occlusion times. A subset of animals (n=5) underwent 60 mins of ischemia resulting in consistent infarct volumes primarily located to the cortex that correlated well with neurologic deficit scores. This approach offers promise for evaluating therapeutic interventions in stroke.

Successful behavioral strategy to unite mother and infant rhesus monkeys (Macaca mulatta) after cesarean delivery.

Developmental studies of pre- to postnatal continuities in rhesus monkeys sometimes require infants be reared with their mothers. However, complications during pregnancy or experimental designs may require cesarean delivery. Owing to lack of published information on this subject, strategies are needed to introduce mothers to their infants following cesarean delivery. Using positive and negative reinforcement techniques we attempted to unite six infant rhesus macaques, Macaca mulatta, to their mothers following c-sections. For our seventh subject, we attempted to cross-foster an infant onto an unrelated female after she had undergone a cesarean surgery for a late-term spontaneous abortion. The mothers varied in age, parity, previous postnatal mothering experience with infants, housing earlier to delivery, and housing subsequent to introduction. Although there were large individual differences among the mother-infant pairs, all seven introductions were successful. The mothers learned to accept and care for their infants from the continuous application of operant conditioning techniques. These data suggest that mother-rearing following cesarean section is a realistic possibility whether required for clinical reasons or for proper experimental control. Furthermore, the ability to successfully mother-rear infants produced from cesarean delivery lessens the impact this potential confound of not being reared by their mothers exerts on many types of developmental studies.

Risk factors associated with surgical site infection and the development of short-term complications in macaques undergoing indwelling vascular access port placement.

BACKGROUND: Risk factors associated with surgical site infection (SSI) and the development of short-term complications in macaques undergoing vascular access port (VAP) placement are evaluated in this study. METHODS: Records from 80 macaques with VAPs were retrospectively reviewed. Logistic regression was used to identify factors associated with short-term post-operative complications. RESULTS: The primary outcome was SSI, which occurred in 21.6% (52.6% in the first 12 months vs. 13% thereafter) of procedures. SSI was associated with major secondary complications including VAP removal (11.4%), wound dehiscence (5.7%), and mechanical catheter occlusion (5.7%). In multivariate modeling, only surgical program progress was a statistically significant predictor of SSI, while animal compliance had a slightly protective effect. CONCLUSIONS: Vascular access ports have a moderate risk of complications, provided the surgical program optimizes best practices. Under complex experimental conditions, VAPs represent an important refinement, both improving animals' overall well-being and environment and reducing stress.