Effects of ketamine analgesia on cardiorespiratory responses and survival to trauma and hemorrhage in rats.

Ketamine is the recommended analgesic on the battlefield for soldiers with hemorrhage, despite a lack of supportive evidence from laboratory or clinical studies. Hence, this study determined the effects of ketamine analgesia on cardiorespiratory responses and survival to moderate (37% blood volume; n = 8/group) or severe hemorrhage (50% blood volume; n = 10/group) after trauma in rats. We used a conscious hemorrhage model with extremity trauma (fibular fracture + soft tissue injury) while measuring mean arterial pressure (MAP), heart rate (HR), and body temperature (Tb) by telemetry, and respiration rate (RR), minute volume (MV), and tidal volume (TV) via whole body plethysmography. Male rats received saline (S) or 5.0 mg/kg ketamine (K) (100 µL/100 g body wt) intra-arterially after trauma and hemorrhage. All rats survived 37% hemorrhage. For 50% hemorrhage, neither survival times [180 min (SD 78) vs. 209 min (SD 66)] nor percent survival (60% vs. 80%) differed between S- and K-treated rats. After 37% hemorrhage, K (compared with S) increased MAP and decreased Tb and MV. After 50% hemorrhage, K (compared with S) increased MAP but decreased HR and MV. K effects on cardiorespiratory function were time dependent, significant but modest, and transient at the analgesic dose given. K effects on Tb were also significant but modest and more prolonged. With the use of this rat model, our data support the use of K as an analgesic in injured, hypovolemic patients.NEW & NOTEWORTHY Ketamine administration at a dose shown to alleviate pain in nonhemorrhaged rats with extremity trauma had only modest and transient effects on multiple aspects of cardiorespiratory function after both moderate (37%) and severe (50%) traumatic hemorrhages. Such effects did not alter survival.

Identifying Plasma Derived Extracellular Vesicle (EV) Contained Biomarkers in the Development of Chronic Neuropathic Pain.

Research into potentially novel biomarkers for chronic pain development is lacking. microRNAs (miRNAs) are attractive candidates as biomarkers due to their conservation across species, stability in liquid biopsies, and variation that corresponds to a pathologic state. miRNAs can be sorted into extracellular vesicles (EVs) within the cell and released from the site of injury. EVs transfer cargo molecules between cells thus affecting key intercellular signaling pathways. The focus of this study was to determine the plasma derived EV miRNA content in a chronic neuropathic pain rat model. This was accomplished by performing either spinal nerve ligation (SNL; n = 6) or sham (n = 6) surgery on anesthetized male Sprague-Dawley rats. Mechanosensitivity was assessed and plasma derived EV RNA was isolated at baseline (BL), day 3, and 15 postnerve injury. EV extracted small RNA was sequenced followed by differentially expressed (DE) miRNAs and gene target enrichment/signaling pathway analysis performed using R packages and TargetScan/Ingenuity pathway analysis (IPA), respectively. Seven of the DE miRNAs were validated by Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR). The data indicated that SNL rats displayed a time-dependent threshold reduction in response to evoked stimuli from day 3 to day 15 postnerve injury. The data also revealed that 22 and 74 miRNAs at day 3 and 15, respectively, and 33 miRNAs at both day 3 and 15 were uniquely DE between the SNL and sham groups. The key findings from this proposal include (1) the majority of the DE EV miRNAs, which normally function to suppress inflammation, were downregulated, and (2) several of the plasma derived DE EV miRNAs reflect previously observed changes in the injured L5 nerve. The plasma derived DE EV miRNAs regulate processes important in the development and maintenance of neuropathic pain states and potentially serve as key regulators, biomarkers, and targets in the progression and treatment of chronic neuropathic pain. PERSPECTIVE: This article describes the DE miRNA content of plasma derived EVs, comparing neuropathic pain to normal conditions. This data indicates that EV miRNAs may be important in nociception and may also serve as biomarkers for chronic pain. These results encourage further research on EV miRNAs in chronic neuropathic pain sufferers.

Involvement of brain-derived neurotrophic factor (BDNF) in chronic intermittent stress-induced enhanced mechanical allodynia in a rat model of burn pain.

BACKGROUND: Reports show that stressful events before injury exacerbates post-injury pain. The mechanism underlying stress-induced heightened thermal pain is unclear. Here, we examined the effects of chronic intermittent stress (CIS) on nociceptive behaviors and brain-derived nerve growth factor (BDNF) system in the prefrontal cortex (PFC) and hypothalamus of rats with and without thermal injury. RESULTS: Unstressed rats showed transient mechanical allodynia during stress exposure. Stressed rats with thermal injury displayed persistent exacerbated mechanical allodynia (P < 0.001). Increased expression of BDNF mRNA in the PFC (P < 0.05), and elevated TrkB and p-TrkB (P < 0.05) protein levels in the hypothalamus were observed in stressed rats with thermal injury but not in stressed or thermally injured rats alone. Furthermore, administration of CTX-B significantly reduced stress-induced exacerbated mechanical allodynia in thermally injured rats (P < 0.001). CONCLUSION: These results indicate that BDNF-TrkB signaling in PFC and hypothalamus contributes to CIS-induced exacerbated mechanical allodynia in thermal injury state.

Relatively enriched housing conditions delay binge onset but do not attenuate binge size.

Housing and enrichment conditions are essential factors to consider when using animal models of behavior, as they can alter the behavior that is under investigation. The goal of this study was to determine the impact of the relatively enriched environment recommended by current animal care guidelines on development and maintenance of binge-type behavior in rats, using the limited access (LA) binge model. Non-food-deprived rats were divided into two groups, enriched and nonenriched, with all rats housed in shoebox cages. Bedding, nesting material, toys, and a solid floor were provided only to the enriched group to create a state of relative enrichment, or RE, compared to the nonenriched conditions historically used in the LA model. Enriched and nonenriched groups were further divided into control and experimental groups. Control rats received access to an optional source of fat (vegetable shortening) for 30min each day (daily access) while experimental rats received 30-min optional fat access on Monday, Wednesday, and Friday only (intermittent access). The four groups were designated C-E (Control-Enriched), C-NE (Control-Nonenriched), I-E (Intermittent-Enriched), and I-NE (Intermittent-Nonenriched). Bingeing in the LA model is established when a group with intermittent access (i.e., the I-E or I-NE group) consumes significantly more vegetable shortening during the limited access period than a group with daily access (i.e., the C-E or C-NE group). Access sessions continued for 8weeks under these conditions, at which time the housing conditions of the I-E and I-NE groups were reversed for an additional 8weeks of access sessions. Intakes of the C-E and C-NE groups were similar and data from these two groups were combined. Relative to this Combined Control Group (CCG), the I-NE group began bingeing in week 3 while the I-E group binged during weeks 6 and 8. Following the reversal at the beginning of week 9, the newly enriched I-NE group ceased bingeing in week 9 but resumed bingeing in weeks 10-16. The newly nonenriched I-E group continued bingeing through the remainder of the study. Intakes of the I-E and I-NE groups were not significantly different at any time during the study. These results indicate that RE delays binge onset; that is, RE increases the time between the first fat access session and the first occurrence of bingeing. However, RE does not significantly alter the amount of fat consumed during binge sessions. Furthermore, addition of RE to a nonenriched group of animals (I-NE) does not reverse established binge behavior. Thus it appears that regardless of enrichment condition, intermittent access to vegetable shortening induces greater consumption of fat than does daily access. However, it is clear that a certain level of austerity in housing conditions is required for rapid development of lasting binge-type eating to occur. In addition, results suggest that it is unlikely that enrichment, to the degree provided in this study, can prevent or reverse binge-type eating in rats.

Increased Expression of Pyloric ERß Is Associated With Diabetic Gastroparesis in Streptozotocin-Induced Male Diabetic Rats.

BACKGROUND: Gastroparesis is a significant co-morbidity affecting up to 50% of patients with diabetes and is disproportionately found in women. Prior studies have suggested that loss of interstitial cells of Cajal, hyperglycemia, and nitric oxide dysfunction are potential causes of gastroparesis. Since diabetic gastroparesis affects more women than men, we performed an exploratory study with a diabetic rat model to determine if sex hormone signaling is altered in those where gastroparesis develops. METHODS: We injected male rats with streptozotocin (STZ) to model type I diabetes, as confirmed by blood glucose levels. Gastroparesis was determined by acetaminophen gavage and serum acetaminophen levels. Rats were grouped based on acetaminophen and blood glucose data: diabetic (DM), diabetic and gastroparetic (DM + GP), and control (CM). Serum levels of testosterone, estrogen, and insulin were determined as well as aromatase expression in pyloric tissue and serum. Androgen receptor and estrogen receptor α (ERα) and ß (ERß) were also measured in the pylorus. RESULTS: Compared to CM, estrogen increased and testosterone decreased in both DM and DM + GP rats. Sex hormone levels were not different between DM and DM + GP. Serum aromatase was increased in DM and DM + GP rats; however, pyloric tissue levels were not significantly different from controls. ERα was unchanged and androgen receptor decreased in DM and DM + GP. ERß was increased only in DM + GP animals. CONCLUSION: Our study implicates increased pyloric ERß in the development of gastroparesis in STZ-induced male diabetic rats. Increased serum aromatase is likely responsible for altered sex hormone levels. Our study supports the implication of sex hormone signaling in diabetic development and demonstrates a potential unique role for pyloric ERß in male diabetic gastroparesis.

High-throughput vector-borne disease environmental surveillance by polymerase chain reaction according to international accreditation requirements.

Although vector-borne diseases are specific to the region of the host, there is a necessity for surveillance or reference laboratories to perform standardized, high-throughput testing capable of meeting the needs of a changing military environment and response efforts. The development of standardized, high-throughput, semiquantitative real-time and reverse transcription real-time polymerase chain reaction (PCR) methods allows for the timely dissemination of data to interested parties while providing a platform in which long-term sample storage is possible for the testing of new pathogens of interest using a historical perspective. PCR testing allows for the analysis of multiple pathogens from the same sample, thus reducing the workload of entomologists in the field and increasing the ability to determine if a pathogen has spread beyond traditionally defined locations. US Army Public Health Command Region-Europe (USAPHCR-Europe) Laboratory Sciences (LS) has standardized tests for 9 pathogens at multiple life stages. All tests are currently under international accreditation standards. Using these PCR methods and laboratory model, which have universal Department of Defense application, the USAPHCR-Europe LS will generate quality data that is scientifically sound and legally defensible to support force health protection for the US military in both deployed and garrison environments.

Transgenic rescue of ataxia mice reveals a male-specific sterility defect.

Homozygous ataxia (ax(J)) mice have reduced expression of ubiquitin-specific protease 14 (Usp14), resulting in severe neuromuscular defects and death by 2 months of age. Transgenic expression of Usp14 exclusively in the nervous system of ax(J) mice (ax(J)-Tg) prevents early lethality and restores motor system function to the ax(J) mice, enabling an analysis of the reproductive capabilities of Usp14-deficient mice. Although female ax(J)-Tg mice had a 75% reduction of Usp14 in the ovaries, they were able to produce normal litters. Ovary transfer experiments also demonstrated that the ovaries of ax(J) mice were capable of producing viable pups. In contrast, male ax(J) and ax(J)-Tg mice displayed a 50% reduction in testicular Usp14 levels and were infertile, indicating that Usp14 is required for development and function of the male reproductive system. Immunohistochemistry experiments showed that Usp14 is found in the redundant nuclear envelope and cytoplasmic droplet of epididymal spermatozoa. Analysis of ax(J) testes demonstrated a 50% reduction in testis weight, a 100-fold reduction in sperm number and the presence of abnormal spermatozoa in the epididymis. Histological examination of the Usp14-deficient testes revealed abnormal spermatogenesis and the presence of degenerating germ cells, indicating that Usp14 and the ubiquitin proteasome system are required for spermatid differentiation during spermiogenesis.

Lysosomal enzyme cathepsin D protects against alpha-synuclein aggregation and toxicity.

α-synuclein (α-syn) is a main component of Lewy bodies (LB) that occur in many neurodegenerative diseases, including Parkinson's disease (PD), dementia with LB (DLB) and multi-system atrophy. α-syn mutations or amplifications are responsible for a subset of autosomal dominant familial PD cases, and overexpression causes neurodegeneration and motor disturbances in animals. To investigate mechanisms for α-syn accumulation and toxicity, we studied a mouse model of lysosomal enzyme cathepsin D (CD) deficiency, and found extensive accumulation of endogenous α-syn in neurons without overabundance of α-syn mRNA. In addition to impaired macroautophagy, CD deficiency reduced proteasome activity, suggesting an essential role for lysosomal CD function in regulating multiple proteolytic pathways that are important for α-syn metabolism. Conversely, CD overexpression reduces α-syn aggregation and is neuroprotective against α-syn overexpression-induced cell death in vitro. In a C. elegans model, CD deficiency exacerbates α-syn accumulation while its overexpression is protective against α-syn-induced dopaminergic neurodegeneration. Mutated CD with diminished enzymatic activity or overexpression of cathepsins B (CB) or L (CL) is not protective in the worm model, indicating a unique requirement for enzymatically active CD. Our data identify a conserved CD function in α-syn degradation and identify CD as a novel target for LB disease therapeutics.

Transgenic rescue of ataxia mice with neuronal-specific expression of ubiquitin-specific protease 14.

The ataxia mutation (axJ) is a recessive neurological mutation that results in reduced growth, ataxia, and hindlimb muscle wasting in mice. The axJ gene encodes ubiquitin-specific protease 14 (Usp14), a deubiquitinating enzyme (DUB) that associates with the proteasome via its ubiquitin-like (Ubl) domain and is involved in processing ubiquitin chains. Analysis of Usp14 gene products demonstrated that Usp14 undergoes alternative pre-mRNA splicing to produce a full-length form of Usp14 that is capable of binding proteasomes and a form that contains a deletion in the Ubl domain. The full-length form of Usp14 is the only form that appears to be reduced in the axJ mice. Transgenic rescue of the axJ mice with neuronal-specific expression of Usp14 demonstrated that the full-length form of Usp14 was sufficient to restore viability and motor system function to the axJ mice. Biochemical analysis showed that the ubiquitin hydrolyase activity of this form of Usp14 is dependent on the presence of proteasomes, and neuronal expression of full-length Usp14 was able to restore the levels of monomeric ubiquitin in the brains of axJ mice. However, the axJ-rescued mice still displayed the Purkinje cell axonal swellings that are seen in the axJ mice, indicating that this cerebellar alteration is not the primary cause of the axJ movement disorders. These results show that the motor defects observed in the axJ mice are attributable to a neuropathic disease rather than to a muscular disorder and suggest that changes in proteasomal function may contribute to neurological dysfunction in the axJ mice.

Loss of Usp14 results in reduced levels of ubiquitin in ataxia mice.

The ataxia (ax(J)) mutation is a spontaneous recessive mutation that results in reduced expression of ubiquitin-specific protease 14, Usp14. Mice homozygous for the ax(J) mutation are retarded for growth and exhibit several behavioral disorders, including a resting tremor and hindlimb paralysis. Although pathological defects appear to be limited to the central nervous system, reduction of Usp14 expression was widespread in the ax(J) mice. Usp14 co-fractionated with proteasomes isolated from livers and brains of wild-type mice. Proteasomes isolated from the ax(J) brains still possessed deubiquitinating activity and were functionally competent to hydrolyze 20S proteasomal substrates in vitro. However, the levels of monomeric ubiquitin were reduced approximately 35% in most of the ax(J) tissues examined. These results indicate that Usp14 functions to maintain the cellular levels of monomeric ubiquitin in mammalian cells, and that alterations in the levels of ubiquitin may contribute to neurological disease.