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Item Evaluation of the Aesthetics of Physical Methods of Euthanasia of Anesthetized Rats(American Association for Laboratory Animal Science, 2011-09) Hickman, Debra L.; Johnson, Steven W.; Laboratory Animal Resource Center, IU School of MedicineDissection of living brain tissue for in vitro experiments requires the use of a rapid euthanasia method. However, the method must not subject animals to unnecessary pain and must be aesthetically acceptable to experimenters. The purposes of the current study were to assess the aesthetics of 6 euthanasia methods, measure the procedure duration, and evaluate brain for pathology after each procedure. We digitally recorded euthanasia of isoflurane-anesthetized rats by 6 physical methods: anesthetic overdose, cardiac exsanguination, decapitation, closed intrathoracic transection of the great vessels and heart, thoracic percussion, and thoracotomy with rupture of great vessels. Volunteer researchers and animal caretakers watched the video and completed an associated questionnaire. Anesthetic overdose and cardiac exsanguinations were rated most aesthetically pleasing, although these procedures took the longest to complete. In contrast, decapitation and thoracic percussion were the least aesthetically pleasing, but these methods were the quickest. No demographic factor was identified that could predict whether a given euthanasia procedure would be favored for aesthetic reasons, and participants provided a wide variety of rationales for the aesthetic ratings they assigned. Although all of these euthanasia methods meet the criteria of approved methods of euthanasia of anesthetized rats as defined by the AVMA, aesthetic features and the scientific need for rapid euthanasia are both considerations in selecting a method.Item IGF-I Receptor Localization and Constant Infusion of a Supraphysiologic Dose of IGF-I in the Sprague-Dawley Rat(1993) Alford, Timothy J.; Simmons, Kirt; Roberts, W. Eugene; Garetto, Lawrence P.; Hughes, Christopher; Bixler, DavidPrevious studies have shown an increased growth of the tibial growth plate in rats infused with supraphysiologic doses of IGF-I. However, no one has demonstrated this effect on the TMJ in vivo. To determine the effect of a constant infusion of IGF-1 on the TMJ, 20 Sprague-Dawley rats were divided into three groups: (1) control, (2) surgical control, and (3) IGF-1 and placebo infused. IGF-I was delivered at a rate of 1 μg/day over the TMJ via osmotic minipumps. lntravital bone labels were administered at two-week intervals to monitor growth rate. Following sacrifice, seven mandibular (Mn) dimensions were measured anthropometrically. The mandibles were then imbedded in acrylic and stained with tetrachrome to visualize the Mn cartilage. Fluorescence microscopy was utilized to measure the Mn growth between bone labels and calculate growth rates. In addition, the hypertrophic cartilage layer thickness was measured photomicrographically. ANOVA showed no significant difference (P<0.05) in growth rates or cartilage thicknesses between the groups. However, multiple t tests did show an increase in several Mn dimensions (increase in length from gonion to the mental foramen; increase in length from condylion to the mental foramen; and increase in condylar head anterior-posterior length) in the experimental animals comparing the IGF-I infused side with the placebo infused control side. Therefore, it was concluded that IGF-I, when infused at a constant supraphysiologic dose, may increase mandibular growth in certain directions. The present study is not able to definitively demonstrate that these increases are due to direct effects on Mn cartilage growth.Item Improving the Patency of Jugular Vein Catheters in Sprague-Dawley Rats by Using an Antiseptic Nitrocellulose Coating(Ingenta, 2018-09-01) De Luca, Thomas; Szilágyi, Keely L; Hargreaves, Katherine A.; Collins, Kimberly S.; Benson, Eric A.; Department of Medicine, Indiana University School of MedicinePreclinical studies in animals often require frequent blood sampling over prolonged periods. A preferred method in rats is the implantation of a polyurethane catheter into the jugular vein, with heparinized glycerol as a lock solution. However, analysis of various biologic compounds (for example, microRNA) precludes the use of heparin. We used sodium citrate as an alternative to heparin but observed more frequent loss of catheter patency. We hypothesized that this effect was due to evaporation of lock solution at the exteriorized portion of the catheter, subsequent blood infiltration into the catheter, and ultimately clot formation within the catheter. We therefore tested evaporation and its variables in vitro by using 5 common catheter materials. We used the migration of dye into vertically anchored catheters as a measure of lock displacement due to evaporation. Exposure to dry room-temperature air was sufficient to cause dye migration against gravity, whereas a humid environment and adding glycerol to the lock solution mitigated this effect, thus confirming loss of the lock solution from the catheter by evaporation. We tested 4 catheter treatments for the ability to reduce lock evaporation. Results were validated in vivo by using male Sprague-Dawley rats (n = 12) implanted with polyurethane jugular vein catheters and randomized to receive a nitrocellulose-based coating on the exteriorized portion of the catheter. Coating the catheters significantly improved patency, as indicated by a Kaplan-Meier log-rank hazard ratio greater than 5 in untreated catheters. We here demonstrate that a simple nitrocellulose coating reduces evaporation from and thus prolongs the patency of polyurethane catheters in rats.Item Induction of chronic migraine phenotypes in a rat model after environmental irritant exposure(Lippincott, Williams & Wilkins, 2018-03) Kunkler, Phillip Edward; Zhang, LuJuan; Johnson, Philip Lee; Oxford, Gerry Stephen; Hurley, Joyce Harts; Biochemistry and Molecular Biology, School of MedicineAir pollution is linked to increased emergency department visits for headache and migraine patients frequently cite chemicals or odors as headache triggers, but the association between air pollutants and headache is not well understood. We previously reported that chronic environmental irritant exposure sensitizes the trigeminovascular system response to nasal administration of environmental irritants. Here, we examine whether chronic environmental irritant exposure induces migraine behavioral phenotypes. Male rats were exposed to acrolein, a transient receptor potential channel ankyrin-1 (TRPA1) agonist, or room air by inhalation for 4 days before meningeal blood flow measurements, periorbital cutaneous sensory testing, or other behavioral testing. Touch-induced c-Fos expression in trigeminal nucleus caudalis was compared in animals exposed to room air or acrolein. Spontaneous behavior and olfactory discrimination was examined in open-field testing. Acrolein inhalation exposure produced long-lasting potentiation of blood flow responses to a subsequent TRPA1 agonist and sensitized cutaneous responses to mechanical stimulation. C-Fos expression in response to touch was increased in trigeminal nucleus caudalis in animals exposed to acrolein compared with room air. Spontaneous activity in an open-field and scent preference behavior was different in acrolein-exposed compared with room air-exposed animals. Sumatriptan, an acute migraine treatment blocked acute blood flow changes in response to TRPA1 or transient receptor potential vanilloid receptor-1 agonists. Pretreatment with valproic acid, a prophylactic migraine treatment, attenuated the enhanced blood flow responses observed after acrolein inhalation exposures. Environmental irritant exposure yields an animal model of chronic migraine in which to study mechanisms for enhanced headache susceptibility after chemical exposure.Item Inhibition of the Ubc9 E2 SUMO-conjugating enzyme-CRMP2 interaction decreases NaV1.7 currents and reverses experimental neuropathic pain(Lippincott, Williams & Wilkins, 2018-10) François-Moutal, Liberty; Dustrude, Erik T.; Wang, Yue; Brustovetsky, Tatiana; Dorame, Angie; Ju, Weina; Moutal, Aubin; Perez-Miller, Samantha; Brustovetsky, Nickolay; Gokhale, Vijay; Khanna, May; Khanna, Rajesh; Pharmacology and Toxicology, School of MedicineWe previously reported that destruction of the small ubiquitin-like modifier (SUMO) modification site in the axonal collapsin response mediator protein 2 (CRMP2) was sufficient to selectively decrease trafficking of the voltage-gated sodium channel NaV1.7 and reverse neuropathic pain. Here, we further interrogate the biophysical nature of the interaction between CRMP2 and the SUMOylation machinery, and test the hypothesis that a rationally designed CRMP2 SUMOylation motif (CSM) peptide can interrupt E2 SUMO-conjugating enzyme Ubc9-dependent modification of CRMP2 leading to a similar suppression of NaV1.7 currents. Microscale thermophoresis and amplified luminescent proximity homogeneous alpha assay revealed a low micromolar binding affinity between CRMP2 and Ubc9. A heptamer peptide harboring CRMP2's SUMO motif, also bound with similar affinity to Ubc9, disrupted the CRMP2-Ubc9 interaction in a concentration-dependent manner. Importantly, incubation of a tat-conjugated cell-penetrating peptide (t-CSM) decreased sodium currents, predominantly NaV1.7, in a model neuronal cell line. Dialysis of t-CSM peptide reduced CRMP2 SUMOylation and blocked surface trafficking of NaV1.7 in rat sensory neurons. Fluorescence dye-based imaging in rat sensory neurons demonstrated inhibition of sodium influx in the presence of t-CSM peptide; by contrast, calcium influx was unaffected. Finally, t-CSM effectively reversed persistent mechanical and thermal hypersensitivity induced by a spinal nerve injury, a model of neuropathic pain. Structural modeling has now identified a pocket-harboring CRMP2's SUMOylation motif that, when targeted through computational screening of ligands/molecules, is expected to identify small molecules that will biochemically and functionally target CRMP2's SUMOylation to reduce NaV1.7 currents and reverse neuropathic pain.Item Interstrain differences in the development of pyometra after estrogen treatment of rats(American Association for Laboratory Animal Science, 2009-09) Brossia, Lisa Jane; Roberts, Christopher Sean; Lopez, Jennifer T.; Bigsby, Robert M.; Dynlacht, Joseph R.; Pharmacology and Toxicology, School of MedicineThis case report describes the unanticipated development of pyometra in Brown Norway rats after treatment with estrogen. Sprague Dawley and Brown Norway rats were ovariectomized and randomly assigned to treatment groups (subcutaneous implantation of either a capsule containing 20 mg 17beta-estradiol or an empty capsule, as a control). After irradiation of only the right eye, the rats were followed for several months in an attempt to determine the effects of estrogen on radiation cataractogenesis and investigate potential strain differences in this phenomenon. However, all Brown Norway rats that received estradiol treatment developed pyometra, whereas none the Sprague Dawley or control Brown Norway rats did. This case demonstrates the potential adverse effects of exogenous estrogen therapy, which are strain-specific in the rat. Caution should be taken when designing estrogen-related experiments involving Brown Norway rats and other potentially sensitive strains.Item Morphologic Changes in the Mandibular Condyle of Growing Sprague-Dawley Rats After Electrolytic Lesioning of the Trigeminal Motor Nucleus(1994) Hurst, Charles A.; Byrd, Kenneth E.; Roberts, W. Eugene; Garetto, Lawrence P.; Hohlt, William; Burr, DavidLesioning motoneurons in the brainstem alters biomechanical forces and affects craniofacial growth by producing skeletal asymmetries. The purpose of this study was to examine changes that occur in the mandibular condyle in rats that have had their trigeminal motor nucleus (TMN) lesioned. The following null hypothesis was tested: unilateral electrolytic lesioning of the TMN has no effect on condylar morphology in growing rats. To accomplish this, experimental rats received a small electrolytic lesion in their left side TMN. The controls received a sham lesion that caused TMN stimulation with no electrolytic lesion produced. Seven rats from each group were sacrificed at 28, 56, and 84 days postoperatively. The rats were decapitated and their skulls were dried. Mandibular condyles were harvested from the dry rat skulls. The specimens were embedded and sectioned. The sections were stained with H&E. The following parameters were measured: condyle perimeter, condylar widths at 125 μm increments measured with a grid aligned with the condylar neck, width of the condylar neck, and bone surface area proximal to the condylar neck measurement. Experimental groups were compared with control groups by means of factorial analysis of variance, ANOVA, with the factors being the experimental operation and the time of sacrifice. Findings show significant or near borderline significant F tests for right-left differences and side-by-group interactions for width at 625 μm, 750 μm, 875 μm, and 1000 μm from the top of the condyle; but not at the other widths measured. Right-left difference and side-by-time interaction for shape factor measurement were also shown to be significant. The null hypothesis stating unilateral electrolytic lesioning of the TMN has no effect on condylar morphology in growing rats was therefore rejected. The failure to reach significance in some parameters may have been due to the small number of specimens. Due to the fragile nature of the dried specimens, group numbers ranged from seven to four condyle pairs per group. In conclusion, lesions to the TMN of growing rats affect the morphology of the mandibular condyle in the medial-lateral plane. Alterations in morphology during growth after lesioning the TMN were likely caused by changes in the neuromuscular activity of masticatory muscles and their biomechanical effects on bone. Data in this study suggest that it is valuable to view mandibular condyles from a frontal view (i.e., frontal tomography) when altered condylar morphology in human patients is suspected.Item Muscimol acts in dorsomedial but not paraventricular hypothalamic nucleus to suppress cardiovascular effects of stress(Society for Neuroscience, 1996-02-01) Stotz-Potter, E. H.; Willis, L. R.; DiMicco, J. A.; Pharmacology and Toxicology, School of MedicineBoth the dorsomedial hypothalamic nucleus (DMH) and the paraventricular hypothalamic nucleus (PVN) have been implicated in the neural control of the cardiovascular response to stress. We used the GABAA agonist muscimol to inhibit neuronal activation and attempted to identify hypothalamic nuclei required for the cardiovascular response to air stress. Chronically instrumented rats received bilateral injections of either 80 pmol of muscimol or 100 nl of saline vehicle into the DMH, the PVN, or an intermediate area (including the rostral edge of the DMH and the region between the two nuclei) and were placed immediately in a restraining tube and subjected to 20 min of air stress. In all rats, air stress after vehicle injections caused marked increases in heart rate (137 +/- 6 beats/min) and blood pressure (26 +/- 2 mmHg). Microinjection of muscimol into the DMH suppressed the heart rate and blood pressure response by 85 and 68%, respectively. Identical microinjection of muscimol into the intermediate area between the DMH and the PVN attenuated the increases in heart rate by only 46% and in blood pressure by 52%. In contrast, similar injections into the vicinity of the PVN failed to alter the cardiovascular response to air stress. These findings demonstrate that muscimol-induced inhibition of neuronal activity in the region of the DMH blocks air stress-induced increases in heart rate and arterial pressure, whereas similar treatment in the area of the PVN has no effect.Item Prostaglandin E2 enhances bradykinin-stimulated release of neuropeptides from rat sensory neurons in culture(Society for Neuroscience, 1994-08) Vasko, MR; Campbell, WB; Waite, KJ; Pharmacology and Toxicology, School of MedicineProstaglandins are known to enhance the inflammatory and nociceptive actions of other chemical mediators of inflammation such as bradykinin. One possible mechanism for this sensitizing action is that prostanoids augment the release of neuroactive substances from sensory neurons. To initially test this hypothesis, we examined whether selected prostaglandins could enhance the resting or bradykinin-evoked release of immunoreactive substance P (iSP) and/or immunoreactive calcitonin gene-related peptide (iCGRP) from sensory neurons in culture. Bradykinin alone causes a concentration-dependent increase in the release of iSP and iCGRP from isolated sensory neurons, and this action is abolished in the absence of extracellular calcium. Pretreating the neurons with PGE2 (10 nM to 1 microM) potentiates the bradykinin-evoked release of both iSP and iCGRP by approximately two-to fourfold. At these concentrations, PGE2 alone did not significantly alter peptide release. Exposing the cultures to 1 microM PGF2 alpha is ineffective in altering either resting or bradykinin-evoked peptide release. Sensory neurons in culture contain cyclooxygenase-like immunoreactivity suggesting that the enzyme that converts arachidonic acid to prostaglandins is present. In addition, pretreating cultures with 14C-arachidonic acid yields radiolabeled eicosanoids that cochromatograph with known prostaglandin standards. Preexposing cultures to indomethacin abolishes the production of prostaglandins and attenuates the bradykinin-stimulated release of iSP and iCGRP. This implies that the synthesis of prostaglandins contributes to the bradykinin-evoked release of peptides. The augmentation of bradykinin-induced release of iSP and iCGRP by PGE2 may be one mechanism to account for the inflammatory and hyperalgesic actions of this eicosanoid.Item Prostaglandins facilitate peptide release from rat sensory neurons by activating the adenosine 3',5'-cyclic monophosphate transduction cascade(Society for Neuroscience, 1995-07) Hingtgen, C.M.; Waite, K.J.; Vasko, M.R.; Pharmacology and Toxicology, School of MedicineProstaglandins sensitize sensory neurons to activation by mechanical, thermal and chemical stimuli. This sensitization also results in an increase in the stimulus-evoked release of the neuroactive peptides, substance P and calcitonin gene-related peptide from sensory neurons. The cellular transduction cascade underlying the prostaglandin-induced augmentation of peptide release is not known. Therefore, we examined whether the sensitizing action of prostaglandins on peptide release from sensory neurons grown in culture is mediated by the second messenger, adenosine 3', 5' cyclic monophosphate (cAMP). Prostaglandin E2 and carba prostacyclin (a stable analog of prostaglandin I2) significantly increase the content of cAMP-like immunoreactive substance (icAMP) in the sensory neuron cultures at concentrations that also augment the bradykinin- or capsaicin-evoked release of peptides. Furthermore, pretreating sensory neurons with agents that increase intracellular cAMP mimics the sensitizing action of prostaglandins. Exposing cultures to either forskolin (0.1-10 microM), cholera toxin (1.5 micrograms), or 8-bromo-cAMP (100 microM) results in a significant enhancement of the bradykinin- or capsaicin-stimulated release of both substance P-like and calcitonin gene-related peptide-like immunoreactive substances. Pretreating sensory neurons with the adenylyl cyclase inhibitor, 9-tetrahydro-2-furyl adenine (5 mM), abolishes the prostaglandin-induced increases in icAMP content and attenuates the prostaglandin E2 or carba prostacyclin enhancement of the evoked release of calcitonin gene-related peptide-like immunoreactive substance. These results demonstrate that the cAMP transduction cascade mediates the sensitizing actions of prostaglandins on peptide release from sensory neurons.