Heidi L. Lujan

Reduced arsenic clearance and increased toxicity in aquaglyceroporin-9-null mice

Expressed in liver, aquaglyceroporin-9 (AQP9) is permeated by glycerol, arsenite, and other small, neutral solutes. To evaluate a possible protective role, AQP9-null mice were evaluated for in vivo arsenic toxicity. After injection with NaAsO2, AQP9-null mice suffer reduced survival rates (LD50, 12 mg/kg) compared with WT mice (LD50, 15 mg/kg). The highest tissue level of arsenic is in heart, with AQP9-null mice accumulating 10–20 times more arsenic than WT mice. Within hours after NaAsO2 injection, AQP9-null mice sustain profound bradycardia, despite normal serum electrolytes. Increased arsenic levels are also present in liver, lung, spleen, and testis of AQP9-null mice. Arsenic levels in the feces and urine of AQP9-null mice are only ≈10% of the WT levels, and reduced clearance of multiple arsenic species by the AQP9-null mice suggests that AQP9 is involved in the export of multiple forms of arsenic. Immunohistochemical staining of liver sections revealed that AQP9 is most abundant in basolateral membrane of hepatocytes adjacent to the sinusoids. AQP9 is not detected in heart or kidney by PCR or immunohistochemistry. We propose that AQP9 provides a route for excretion of arsenic by the liver, thereby providing partial protection of the whole animal from arsenic toxicity.

VGLUT1 and VGLUT2 innervation in autonomic regions of intact and transected rat spinal cord

Fast excitatory neurotransmission to sympathetic and parasympathetic preganglionic neurons (SPN and PPN) is glutamatergic. To characterize this innervation in spinal autonomic regions, we localized immunoreactivity for vesicular glutamate transporters (VGLUTs) 1 and 2 in intact cords and after upper thoracic complete transections. Preganglionic neurons were retrogradely labeled by intraperitoneal Fluoro‐Gold or with cholera toxin B (CTB) from superior cervical, celiac, or major pelvic ganglia or adrenal medulla. Glutamatergic somata were localized with in situ hybridization for VGLUT mRNA. In intact cords, all autonomic areas contained abundant VGLUT2‐immunoreactive axons and synapses. CTB‐immunoreactive SPN and PPN received many close appositions from VGLUT2‐immunoreactive axons. VGLUT2‐immunoreactive synapses occurred on Fluoro‐Gold‐labeled SPN. Somata with VGLUT2 mRNA occurred throughout the spinal gray matter. VGLUT2 immunoreactivity was not noticeably affected caudal to a transection. In contrast, in intact cords, VGLUT1‐immunoreactive axons were sparse in the intermediolateral cell column (IML) and lumbosacral parasympathetic nucleus but moderately dense above the central canal. VGLUT1‐immunoreactive close appositions were rare on SPN in the IML and the central autonomic area and on PPN. Transection reduced the density of VGLUT1‐immunoreactive axons in sympathetic subnuclei but increased their density in the parasympathetic nucleus. Neuronal cell bodies with VGLUT1 mRNA occurred only in Clarkes column. These data indicate that SPN and PPN are densely innervated by VGLUT2‐immunoreactive axons, some of which arise from spinal neurons. In contrast, the VGLUT1‐immunoreactive innervation of spinal preganglionic neurons is sparse, and some may arise from supraspinal sources. Increased VGLUT1 immunoreactivity after transection may correlate with increased glutamatergic transmission to PPN. J. Comp. Neurol. 503:741–767, 2007.

A chronic increase in physical activity inhibits fed-state mTOR/S6K1 signaling and reduces IRS-1 serine phosphorylation in rat skeletal muscle

A chronic increase in physical activity and (or) endurance training can improve insulin sensitivity in insulin-resistant skeletal muscle. Cellular mechanisms responsible for the development of insulin resistance are unclear, though one proposed mechanism is that nutrient overload chronically increases available energy, over-activating the mammalian target of rapamycin (mTOR) and ribosomal S6 kinase 1 (S6K1) signaling pathway leading to increased phosphorylation of serine residues on insulin receptor substrate-1 (IRS-1). The objective of this study was to determine if increased physical activity would inhibit mTOR/S6K1 signaling and reduce IRS-1 serine phosphorylation in rat skeletal muscle. Soleus muscle was collected from fed male Sprague-Dawley sedentary rats (Inactive) and rats with free access to running wheels for 9 weeks (Active). Immunoblotting methods were used to measure phosphorylation status of mTOR, S6K1, IRS-1, and PKB/Akt (protein kinase B/AKT), and total abundance of proteins associated with the mTOR pathway. Muscle citrate synthase activity and plasma insulin and glucose concentrations were measured. Phosphorylation of mTOR (Ser2448), S6K1 (Thr389), and IRS-1 (Ser636-639) was reduced in Active rats (p<0.05). Total protein abundance of mTOR, S6K1, IRS-1, 4E-BP1, eEF2, PKB/Akt and AMPKalpha, and phosphorylation of PKB/Akt were unaffected (p>0.05). Total SKAR protein, a downstream target of S6K1, and citrate synthase activity increased in Active rats (p<0.05), though plasma insulin and glucose levels were unchanged (p>0.05). Reduced mTOR/S6K1 signaling during chronic increases in physical activity may play an important regulatory role in the serine phosphorylation of IRS-1, which should be examined as a potential mechanism for attenuation of insulin resistance associated with increased IRS-1 serine phosphorylation.

Dynamic interaction between the heart and its sympathetic innervation following T5 spinal cord transection

Midthoracic spinal cord injury (SCI) is associated with enhanced sympathetic support of heart rate as well as myocardial damage related to calcium overload. The myocardial damage may elicit an enhanced sympathetic support of contractility to maintain ventricular function. In contrast, the level of inotropic drive may be reduced to match the lower afterload that results from the injury-induced reduction in arterial pressure. Accordingly, the inotropic response to midthoracic SCI may be increased or decreased but has not been investigated and therefore remains unknown. Furthermore, the altered ventricular function may be associated with anatomical changes in cardiac sympathetic innervation. To determine the inotropic drive following midthoracic SCI, a telemetry device was used for repeated measurements of left ventricular (LV) function, with and without beta-adrenergic receptor blockade, in rats before and after midthoracic SCI or sham SCI. In addition, NGF content (ELISA) and dendritic arborization (cholera toxin B immunohistochemistry and Sholl analysis) of cardiac-projecting sympathetic postganglionic neurons in the stellate ganglia were determined. Midthoracic SCI was associated with an enhanced sympathetic support of heart rate, dP/dt(+), and dP/dt(-). Importantly, cardiac function was lower following blockade of the sympathetic nervous system in rats with midthoracic SCI compared with sham-operated rats. Finally, these functional neuroplastic changes were associated with an increased NGF content and structural neuroplasticity within the stellate ganglia. Results document impaired LV function with codirectional changes in chronotropic and inotropic responses following midthoracic SCI. These functional changes were associated with a dynamic interaction between the heart and its sympathetic innervation.

Gene and protein expression associated with protein synthesis and breakdown in paraplegic skeletal muscle

Spinal cord injury reduces the rate of skeletal muscle protein synthesis and increases protein breakdown, resulting in rapid muscle loss. The purpose of this study was to determine whether long‐term paraplegia would eventually result in a downregulation of muscle mRNA and protein expression associated with both protein synthesis and breakdown. After 10 weeks of spinal cord transection, soleus muscle from 12 rats (6 sham‐control, 6 paraplegic) was studied for mRNAs and proteins associated with protein synthesis and breakdown using real‐time polymerase chain reaction and immunoblotting techniques. Protein kinase B (PKB/Akt), ribosomal S6 kinase 1 (S6K1), and myogenin mRNA were downregulated, whereas muscle ring finger 1 (MuRF1) and phospho‐forkhead transcription factor 4 (FoxO4) protein were increased in paraplegic rats. We conclude that gene and protein expression of pathways associated with protein synthesis are reduced, whereas some markers of protein breakdown remain elevated following chronic paraplegia. Clinical interventions designed to increase muscle protein synthesis may be helpful in preventing excessive muscle loss during long‐term paraplegia. Muscle Nerve, 2008

Structural neuroplasticity following T5 spinal cord transection: increased cardiac sympathetic innervation density and SPN arborization

When the spinal cord is injured at or below thoracic level 5 (T5), cardiovascular control is markedly unbalanced as the heart and blood vessels innervated by upper thoracic segments remain under brain stem control, whereas the vasculature of the lower body is affected by unregulated spinal reflexes. Importantly, the regulation of heart rate and cardiac function is abnormal after spinal cord injury (SCI) at T5 because sympathetic outflow to the heart is increased. An increase in tonic sympathetic outflow may be attributable to multiple mechanisms, such as increases in cardiac sympathetic innervation density, altered morphology of stellate ganglia neurons, and/or structural neuroplasticity of cardiac sympathetic preganglionic neurons (SPNs). Furthermore, these neuroplastic changes associated with SCI may be mediated by nerve growth factor (NGF). NGF is a neurotrophin that supports the survival and differentiation of sympathetic neurons and enhances target innervation. Therefore, we tested the hypothesis that T5 spinal cord transection (T5X) is associated with an increased left ventricular (LV) NGF content, LV sympathetic innervation density, and cardiac SPN arborization. In intact and paraplegic (9 wk posttransection) rats, LV NGF content (ELISA), LV sympathetic innervation density (tyrosine hydroxylase immunohistochemistry), and cardiac SPN arborization (cholera toxin B immunohistochemistry and Sholl Analysis) were determined. Paraplegia, compared with intact, significantly increased LV NGF content, LV sympathetic innervation density, and cardiac SPN arborization. Thus, altered autonomic behavior following SCI is associated with structural neuroplastic modifications.

Cardiac output, at rest and during exercise, before and during myocardial ischemia, reperfusion, and infarction in conscious mice

Multiple systems and regulatory strategies interact to control cardiac homeostasis. In fact, regulated systems, feedback controls, and redundant control mechanisms dominate in whole animals. Accordingly, molecular and cellular tools and techniques must be utilized in complex models with multiple systems and regulatory strategies to fully appreciate the physiological context. Currently, these techniques are mainly performed under conditions remote from the normal in vivo condition; thus, the extrapolation of molecular changes to the in vivo situation and the facilitation of translational aspect of the findings are limited. A major obstacle has been the reliance on preparations that do not mimic the clinical or physiological situation. This is particularly true regarding measurements of cardiac function in mice. To address these concerns, we used a permanently implanted Doppler ultrasonic flow probe on the ascending aorta and coronary artery occluder for repeated measurements of ascending aortic blood flow (cardiac output) in conscious mice, at rest and during exercise, before and during coronary artery occlusion/reperfusion and infarction. The conscious mouse model permits detailed monitoring of within-animal changes in cardiac function during myocardial ischemia, reperfusion, and infarction in an intact, complex model free of the confounding influences of anesthetics, surgical trauma, and restraint stress. Results from this study suggest that previous protocols may have overestimated resting baseline values and underestimated cardiac output reserve. Using these procedures in currently available spontaneous or engineered mouse mutants has the potential to be of major importance for advancing the concepts and methods that drive cardiovascular research.

Student interaction characteristics during collaborative group testing

We used collaborative testing in a veterinary physiology course (65 students) to answer the following questions: 1) do students with individual correct responses or students with individual incorrect responses change their answers during group testing? and 2) do high-performing students make the decisions, that is, are low-performing students carried by high-performing peers? To address these questions, students first completed the exam in the traditional format as individuals. After completing the exam as individuals, students completed the same exam in groups of two. Finally, the same questions were discussed by the instructor and students (instructor feedback). We found that students with individual incorrect responses changed their answers during group testing more often than students with individual correct responses (odds ratio: 7.58, P < 0.01). Furthermore, student feedback was more beneficial when group members had different individual answers than when they had same individual answers (P < 0.05). In addition, when group members had different individual answers, more answers were changed to correct responses than to incorrect responses (77% vs. 23%, P < 0.01). It was more important to have the correct answer than to be the high-performing student, because the student with the correct response (being either the high- or low-performing student) generally prevailed ( approximately 80% of the time, P = 0.5). Finally, the positive effects of group testing (77% of total effects, P < 0.05) were due to students who changed their individual answer to the correct response after discussion with peers with the correct response and also with the incorrect individual response.

Ventricular function during exercise in mice and rats

The mouse has many advantages over other experimental models for the molecular investigation of left ventricular (LV) function. Accordingly, there is a keen interest in, as well as an intense need for, a conscious, chronically instrumented, freely moving mouse model for the determination of cardiac function. To address this need, we used a telemetry device for repeated measurements of LV function in conscious mice at rest and during exercise. For reference, we compared the responses in mice to the responses in identically instrumented conscious rats. The transmitter body of the telemetry device (rat PA-C40; mouse PA-C10; Data Sciences International, St. Paul, MN) was placed in the intraperitoneal space through a ventral abdominal approach (rat) or subcutaneously on the left flank (mouse). The pressure sensor, located within the tip of a catheter, was inserted into the left ventricle through an apical stab wound (18 gauge for rat; 21 gauge for mouse) for continuous, nontethered, recordings of pulsatile LV pressure. A minimum of 1 wk was allowed for recovery and for the animals to regain their presurgical weight. During the recovery period, the animals were handled, weighed, and acclimatized to the laboratory, treadmill, and investigators. Subsequently, LV parameters were recorded at rest and during a graded exercise test. The results document, for the first time, serial assessment of ventricular function during exercise in conscious mice and rats. This methodology may be adopted for advancing the concepts and ideas that drive cardiovascular research.

Paraplegia increased cardiac NGF content, sympathetic tonus, and the susceptibility to ischemia-induced ventricular tachycardia in conscious rats

Midthoracic spinal cord injury is associated with ventricular arrhythmias that are mediated, in part, by enhanced cardiac sympathetic activity. Furthermore, it is well known that sympathetic neurons have a lifelong requirement for nerve growth factor (NGF). NGF is a neurotrophin that supports the survival and differentiation of sympathetic neurons and enhances target innervation. Therefore, we tested the hypothesis that paraplegia is associated with an increased cardiac NGF content, sympathetic tonus, and susceptibility to ischemia-induced ventricular tachyarrhythmias. Intact and paraplegic (6-9 wk posttransection, T(5) spinal cord transection) rats were instrumented with a radiotelemetry device for recording arterial pressure, temperature, and ECG, and a snare was placed around the left main coronary artery. Following recovery, the susceptibility to ventricular arrhythmias (coronary artery occlusion) was determined in intact and paraplegic rats. In additional groups of matched intact and paraplegic rats, cardiac nerve growth factor content (ELISA) and cardiac sympathetic tonus were determined. Paraplegia, compared with intact, increased cardiac nerve growth factor content (2,146 +/- 286 vs. 180 +/- 36 pg/ml, P < 0.05) and cardiac sympathetic tonus (154 +/- 4 vs. 68 +/- 4 beats/min, P < 0.05) and decreased the ventricular arrhythmia threshold (3.6 +/- 0.2 vs. 4.9 +/- 0.2 min, P < 0.05). Thus altered autonomic behavior increases the susceptibility to ventricular arrhythmias in paraplegic rats.