Methodology for creating a chronic osseointegrated neural interface for prosthetic control in rabbits.

BACKGROUND: Chronic stability and high degrees of selectivity are both essential but somewhat juxtaposed components for creating an implantable bi-directional PNI capable of controlling of a prosthetic limb. While the more invasive implantable electrode arrays provide greater specificity, they are less stable over time due to compliance mismatch with the dynamic soft tissue environment in which the interface is created. NEW METHOD: This paper takes the surgical approach of transposing nerves into bone to create neural interface within the medullary canal of long bones, an osseointegrated neural interface, to provide greater stability for implantable electrodes. In this context, we describe the surgical model for transfemoral amputation with transposition of the sciatic nerve into the medullary canal in rabbits. We investigate the capacity to create a neural interface within the medullary canal histolomorphologically. In a separate proof of concept experiment, we quantify the chronic physiological capacity of transposed nerves to conduct compound nerve action potentials evoked via an Osseointegrated Neural Interface. COMPARISON WITH EXISTING METHOD(S): The rabbit serves as an important animal model for both amputation neuroma and osseointegration research, but is underutilized for the exploration neural interfacing in an amputation setting. RESULTS: Our findings demonstrate that transposed nerves remain stable over 12 weeks. Creating a neural interface within the medullary canal is possible and does not impede nerve regeneration or physiological capacity. CONCLUSIONS: This article represents the first evidence that an Osseointegrated Neural Interface can be surgically created, capable of chronic stimulation/recording from amputated nerves required for future prosthetic control.

Quantification of Collagen Organization after Nerve Repair.

BACKGROUND: Clinical outcomes after nerve injury and repair remain suboptimal. Patients may be plagued by poor functional recovery and painful neuroma at the repair site, characterized by disorganized collagen and sprouting axons. Collagen deposition during wound healing can be intrinsically imaged using second harmonic generation (SHG) microscopy. The purpose of this study was to develop a protocol for SHG imaging of nerves and to assess whether collagen alignment can be quantified after nerve repair. METHODS: Sciatic nerve transection and epineural repair was performed in male rats. The contralateral nerves were used as intra-animal controls. Ten-millimeter nerve segments were harvested and fixed onto slides. SHG images were collected using a 20× objective on a multiphoton microscope. Collagen fiber alignment was calculated using CurveAlign software. Alignment was calculated on a scale from 0 to 1, where 1 represents perfect alignment. Statistical analysis was performed using a linear mixed-effects model. RESULTS: Eight male rats underwent right sciatic nerve repair using 9-0 Nylon suture. There were gross variations in collagen fiber organization in the repaired nerves compared with the controls. Quantitatively, collagen fibers were more aligned in the control nerves (mean alignment 0.754, SE 0.055) than in the repairs (mean alignment 0.413, SE 0.047; P < 0.001). CONCLUSIONS: SHG microscopy can be used to quantitate collagen after nerve repair via fiber alignment. Given that the development of neuroma likely reflects aberrant wound healing, ex vivo and/or in vivo SHG imaging may be useful for further investigation of the variables predisposing to neuroma.

Postoperative Analgesia Provided by Liposomal Hydromorphone in Client-Owned Dogs Undergoing Limb Amputation.

The analgesic efficacy of liposomal hydromorphone (LE-hydro) was tested in dogs undergoing limb amputation. The positive controls (n = 10) received subcutaneous (SQ) hydromorphone (0.2 mg/kg) and 1.5 mL of blank liposomes before surgery; fentanyl continuous rate infusion (CRI), 5-10 µg/kg/hr IV, during and for 24 hr after surgery; and a fentanyl patch at extubation. The negative controls (n = 7) received SQ hydromorphone (0.2 mg/kg) and 1.5 mLs of blank liposomes SQ before surgery, fentanyl CRI (5-10 µg/kg/hr IV) during surgery but stopped at extubation, and a fentanyl patch at extubation. The test group (n = 11) received 3 mg/kg of LE-hydro and 1.5 mL of saline SQ before surgery, 1.5 mL of saline SQ, and a saline CRI during surgery. All groups received a bupivacaine block in the limb prior to amputation and carprofen prior to surgery. Treatment failures, pain scores, opioid side effects, heart rate, respiratory rate, temperature, and client-reported pain and side effects were evaluated. There were three treatment failures in the positive control (3/10) and test groups (3/11). Negative controls had seven treatment failures (7/7). Side effects for all three groups were within expected limits. LE-hydro provides postoperative analgesia equivalent to fentanyl CRI in dogs undergoing limb amputation.

Evaluating the anticancer properties of liposomal copper in a nude xenograft mouse model of human prostate cancer: formulation, in vitro, in vivo, histology and tissue distribution studies.

PURPOSE: Although Cu complexes have been investigated as anticancer agents, there has been no description of Cu itself as a cancer killing agent. A stealth liposomal Cu formulation (LpCu) was studied in vitro and in vivo. METHODS: LpCu was evaluated in prostate cancer origin PC-3 cells by a metabolic cytotoxicity assay, by monitoring ROS, and by flow cytometry. LpCu efficacy was evaluated in vivo using intratumoral and intravenous injections into mice bearing PC-3 xenograft tumors. Toxicology was assessed by performing hematological and blood biochemistry assays, and tissue histology and Cu distribution was investigated by elemental analysis. RESULTS: LpCu and free Cu salts displayed similar levels of cell metabolic toxicity and ROS. Flow cytometry indicated that the mechanisms of cell death were both apoptosis and necrosis. Animals injected i.t. with 3.5 mg/kg or i.v. with 3.5 and 7.0 mg/kg LpCu exhibited significant tumor growth inhibition. Kidney and eye were the main organs affected by Cu-mediated toxicities, but spleen and liver were the major organs of Cu deposition. CONCLUSIONS: LpCu was effective at reducing tumor burden in the xenograft prostate cancer model. There was histological evidence of Cu toxicity in kidneys and eyes of animals treated at the maximum tolerated dose of LpCu 7.0 mg/kg.

A chronic window imaging device for the investigation of in vivo peripheral nerves.

Chronic imaging of the peripheral nervous system with contemporary techniques requires repetitive surgical procedures to reopen an area of interest in order to see underlying biological processes over time. The recurrence of surgical openings on an animal increases trauma, stress, and risk of infection. Such effects can greatly lessen the physiological relevance of any data recorded in this manner. In order to bypass repetitive surgery, a Peripheral Nerve Window (PNW) device has been created for chronic in vivo imaging purposes. Intravital imaging window devices have been used previously to image parts of the rodent model such as the brain, spinal cord, and mammary tissue, but currently have not been used in the peripheral nervous system because of lack of bone anchoring and access to deep nerve tissue. We demonstrate a novel surgical technique in a rat which transposes the sciatic nerve above the surrounding muscle tissue allowing the PNW access to an 8mm section of the nerve. Subsequent days of observation revealed increased vasculature development primarily around the nerve, showing that this preparation can be used to image nerve tissue and surrounding vasculature for up to one week post-implantation.

Evaluation of phospholipid and liposomal S-adenosyl methionine for the treatment of liver injury in a murine model.

We have used a murine model of Acetaminophen induced hepatoxicity to determine if S-adenosyl methionine 1,4 butanedisulfonate (SD4) in liposomes can prevent liver injury when administered immediately prior to acetaminophen, as judged by serum aspartate aminotransferase and alanine aminotransferase levels, and histological evidence of liver necrosis. No protection was observed when mice received 1 g/kg unencapsulated SD4. Partial protection was observed with 5 or 0.5 mg/kg SD4 in unextruded distearoylphosphatidylglycerol (DSPG) liposomes. Protection comparable to that seen in mice receiving encapsulated SD4 is achieved when mice received lipid alone in equivalent amounts, suggesting that the contribution of encapsulated SD4 to the efficacy of the liposomes may be minimal. Unextruded distearoylphosphatidylcholine (DSPC) liposomes show only slight effects even at 50 mg/kg SD4. This is likely caused by the size of unextruded DSPC lipsomes, because extruded DSPC liposomes, whose size is smaller, are of comparable efficacy to unextruded DSPG liposomes.

High-risk pregnancy in rhesus monkeys (Macaca mulatta): a case of ectopic, abdominal pregnancy with birth of a live, term infant, and a case of gestational diabetes complicated by pre-eclampsia.

BACKGROUND: Cases of abdominal pregnancy, in the form of intra-abdominal mummified fetuses, have been described in nonhuman primates. Gestational diabetes and pre-eclampsia are common pregnancy complications in women. METHODS: Two timed-bred rhesus monkeys had high-risk pregnancies, an abdominal pregnancy with delivery of a live term infant, and a case of gestational diabetes that later developed pre-eclampsia. RESULTS: The monkey that had abdominal pregnancy later died from septic peritonitis. The monkey had a colonic adenocarcinoma that may have allowed leakage of intestinal contents into the abdomen. Her infant was fostered to another female and survived. The monkey with gestational diabetes and pre-eclampsia was treated with a regimen similar to that used in women, and a live infant was delivered at day 157 of gestation by Caesarian section. CONCLUSION: These cases underscore the value of timed-breeding and the similarities between pregnancy complications in women and in nonhuman primates.

Liposome dependent delivery of S-adenosyl methionine to cells by liposomes: a potential treatment for liver disease.

The present study demonstrates that the nutritional supplement S-adenosyl methionine (SAMe), the primary methyl donor in mammalian cells, is delivered selectively to cells by anionic liposomes, and is, therefore, a liposome dependent drug. Contrary to our expectations, free SAMe chloride was growth inhibitory in cultured cells. The growth inhibitory potency of SAMe chloride in anionic liposomes composed of distearoylphosphatidylglycerol/cholesterol 2:1 was fivefold greater than that of free SAMe. Neutral liposomes composed of distearoylphosphatidylcholine and cholesterol did not increase the potency of the drug. An improved anionic liposome SAMe formulation was produced by use of the 1,4-butanedisulfonate salt (SD4), adding a metal chelator (EDTA), and lowering the buffer pH from pH 7.0 to pH 4.0. This formulation was 15-fold more potent than free SD4, and was active after more than 28 days at 4 degrees C. SAMe and its potential degradation products were screened for toxicity. Formaldehyde was determined to have potency similar to that of free SAMe chloride in CV1-P cells, suggesting that the growth inhibitory effects of SAMe may partly arise from the formation of formaldehyde. The cytotoxic effects of formaldehyde and the less stable forms of SAMe, (SAMe chloride and SAMe tosylate) were decreased in the presence of 3 mM GSH (IC(50) approximately 0.44 mM). The cytotoxic effects of SD4 were not reduced by GSH, suggesting that this more stable form of SAMe is not toxic through the production of formaldehyde. SD4 in anionic DSPG liposomes stimulated murine IL-6 production in RAW 264 cells at concentrations 25- to 30-fold lower than free drug. This increase in potency for IL-6 production was in keeping with the increase in potency observed in our growth inhibition experiments. These results suggest that SD4 in liposomes may be a potential treatment for acute or chronic liver failure.

A single dose of liposome-encapsulated hydromorphone provides extended analgesia in a rat model of neuropathic pain.

Opioids have been shown to relieve thermal hyperalgesia associated with neuropathic pain. We used a novel technique to produce liposome-encapsulated hydromorphone (LEH), which we then tested in a chronic constriction injury (CCI) thermal hyperalgesia model of neuropathic pain. Rats were divided into sham-operated and CCI groups. Treatments consisted of LEH or standard hydromorphone, administered at surgery or 3 d after surgery, when thermal hyperalgesia had developed in the CCI rats. We measured thermal withdrawal latencies on days 0, 3, and 5. CCI rats given liposome-encapsulated vehicle or standard hydromorphone at surgery developed full thermal hyperalgesia. CCI rats given LEH at surgery exhibited no significant change compared with baseline values in thermal withdrawal latency, indicating that this preparation prevented hyperalgesia after a single injection. CCI rats given LEH on day 3 (that is, after they had developed hyperalgesia) showed reversal of hyperalgesia that persisted to day 5, whereas CCI rats given standard hydromorphone on day 3 showed only brief (approximately 90 min) reversal of hyperalgesia. Preemptive injection of LEH prevented hyperalgesia in this model for as long as 5 d. In addition, hyperalgesia was alleviated for at least 2 d after injection of a single dose of LEH. These results suggest that liposome-encapsulation of hydromorphone offers a convenient and effective means to provide relief from neuropathic pain in this rodent model.

Evaluation of liposome-encapsulated oxymorphone hydrochloride in mice after splenectomy.

The use of mice in biomedical research is increasing, largely due to the production and use of genetically engineered animals. Providing postoperative pain control in mice presents many challenges, and long-acting analgesic preparations would be advantageous for this species. A single subcutaneous injection of a liposome-encapsulated (LE) preparation of oxymorphone was compared with multiple injections of buprenorphine or saline in outbred mice undergoing splenectomy. Control groups were given isoflurane alone or isoflurane and an injection of LE oxymorphone but did not undergo surgery. The following parameters were evaluated for 5 days after surgery and were compared with presurgical baseline data for each group: food and water consumption, body weight, ethographic score, and voluntary exercise on a running wheel. Ethographic scores indicated less postsurgical pain in both groups of mice that received either analgesic preparation compared with mice that received only saline. However, mice given LE oxymorphone had superior postoperative recovery, as measured by wheel-running distance and body weight gain, compared with mice given buprenorphine or saline. Mice undergoing splenectomy had significant decreases in body weight, food and water consumption, voluntary exercise, and other normal behaviors. Administration of liposomal oxymorphone at the time of surgery improved postsurgical recovery as measured by these parameters compared with multiple injections of buprenorphine or saline alone. Administration of LE oxymorphone at the time of surgery improved postsurgical recovery, as measured by these parameters.

Mitochondria in nonalcoholic fatty liver disease.

Nonalcoholic fatty liver (NAFL) is associated with fundamental issues of fat metabolism and insulin resistance. These abnormalities have been linked to impairment of ATP homeostasis, and a growing body of literature has reported mitochondrial abnormalities in various forms of hepatic steatosis. The changes are evident as structural abnormalities, including greatly increased size and the development of crystalline inclusions, and are usually regarded as pathologic, reflecting either a protective or degenerative response to injury. Although the relationships between structural changes,decreased mitochondrial function, and disease states are becoming clearer, the molecular basis for the perturbations is not well understood. Oxidative damage is the most likely causative process and may result in alterations of mitochondrial DNA (mtDNA), stimulated apoptotic pathways, and increased propensity for necrosis.Overall mitochondrial health likely depends on multiple factors including the integrity of the mtDNA, the composition of cellular lipids, lipoprotein trafficking, the balance of pro- and antioxidant factors, and the metabolic demands placed on the liver. Mitochondrial dysfunction may play a role in numerous clinical conditions associated with NAFL, such as hepatocellular carcinoma, lipodystrophy,age-related insulin resistance, gut dysmotility, cryptogenic cirrhosis, a mild form of gaze palsy, and possibly other more severe neurodegenerative diseases. The prominent role of mitochondrial dysfunction in NAFL provides a new and exciting paradigm in which to view this disorder, its complications, and potential dietary and pharmacologic intervention.

Inguinal hernias, endometriosis, and other adverse outcomes in rhesus monkeys following lead exposure.

The Harlow Center for Biological Psychology (HCBP) has a cohort of rhesus monkeys that were exposed to low concentrations of lead acetate in utero or as infants. The lead-exposed animals have been followed for 19 years and have developed four cases of inguinal hernia (males), three cases of endometriosis (females), and one case of immunoblastic lymphoma (male). Retrospective analysis of the data from the original lead-exposed cohort indicates that there is a significant association between lead exposure and the development of inguinal hernia (P=.04). Endometriosis was not significantly associated with lead exposure (P=.36). A case control study also was done to determine the significance of neonatal lead exposure as a risk factor for the development of inguinal hernia and endometriosis. The risk of developing inguinal hernia was significantly increased in lead-exposed animals (OR=20.0, P=.009). The association between endometriosis and lead exposure was also strong (OR=10.13, P<.001). No unmatched variables were associated with inguinal hernia, including body weight, history of diarrhea, constipation, or intussusception. No unmatched variables were highly associated with endometriosis, including body weight, age at first parity, and history of stillbirths. However, parity and the number of stillbirths were associated with lead exposure (P=.011 and P=.041, respectively). There was an association between endometriosis and a history of hysterotomy (OR=2.09) but it was not statistically significant (P=.38). No other cases of lymphoma in unexposed animals were identified using HCBP animal health records. These data indicate that early lead-exposed rhesus monkeys may develop illnesses later in life, especially inguinal hernia and endometriosis, more frequently than unexposed monkeys. Studies of human populations with early lead exposure are warranted to determine their incidence of inguinal hernia, endometriosis, and hematologic neoplasia.

Liposome-encapsulated oxymorphone hydrochloride provides prolonged relief of postsurgical visceral pain in rats.

Adequate pain control is necessary for optimal postsurgical recovery and humane treatment of laboratory and companion animals. Opioid drugs are currently the most potent analgesic agents available in human and veterinary medicine. Long-acting formulations of opioid drugs confer several important advantages over standard pharmaceutical preparations, especially for use in animals. A long-acting formulation of oxymorphone hydrochloride was produced by encapsulation into liposomes. Liposome-encapsulated (LE) oxymorphone was tested in a rat model of visceral postoperative pain. Rats were given one subcutaneous injection of LE oxymorphone (1.2 or 1.6 mg/kg of body weight) or standard oxymorphone (0.3 mg/kg) at the time of intestinal transection or resection. A single administration of LE oxymorphone hydrochloride was as effective for relief of postoperative pain in rats (P = 0.18), as were multiple (q4 h or q8 h) injections of 0.3 mg/kg of the standard pharmaceutical preparation. The rats given LE oxymorphone prior to intestinal resection also had significantly higher body weight at three and seven days after surgery than did rats that were given standard oxymorphone. In conclusion, LE oxymorphone was effective in treating visceral pain associated with intestinal surgery in rats. On the basis of body weight gain, rats treated with LE oxymorphone had improved recovery outcome, compared with rats treated with repeated injections of standard oxymorphone.

A single dose of liposome-encapsulated oxymorphone or morphine provides long-term analgesia in an animal model of neuropathic pain.

An extended-release formulation of oxymorphone was produced by encapsulation into liposomes, using a novel technique. Liposome-encapsulated morphine was produced, using a standard technique These preparations were tested in an animal model of neuropathic pain. Male Sprague-Dawley rats (approx. 300 g) were allotted to control (non-loaded liposomes) and treatment (liposome-encapsulated oxymorphone or morphine) groups. Drugs were administered subcutaneously to all rats immediately prior to sciatic nerve ligation. Thermal withdrawal latencies were measured at baseline and daily for seven days after sciatic nerve ligation. A second experiment involved subcutaneous administration of non-loaded liposomes, morphine, or oxymorphone to rats that did not undergo sciatic nerve ligation. Thermal withdrawal latencies in sciatic nerve-ligated rats given non-loaded liposomes decreased significantly by day four, with maximal decrease at day seven after surgery, indicating development of full hyperalgesia. In contrast, ligated rats given liposome-encapsulated morphine or liposome-encapsulated oxymorphone had no decrease in thermal withdrawal latency by day four, indicating that these long-acting preparations prevented development of hyperalgesia after a single injection. This treatment effect persisted to day seven. Non-ligated rats treated with vehicle or liposome-encapsulated morphine had no change in thermal withdrawal latencies. Non-ligated rats treated with liposome-encapsulated oxymorphone had a small, but significant increase in thermal withdrawal latency from day four through day seven. One subcutaneous injection of liposome-encapsulated oxymorphone or morphine was effective in preventing hyperalgesia in this pain model for up to seven days. These results suggest that liposome-encapsulation of oxymorphone offers a novel, convenient, and effective means to provide long-term analgesia.