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Autism spectrum disorder (ASD) is a social, sensory and developmental condition that affects one in 59 children and specifically one in 42 boys. Despite the 15% increase in prevalence in the last two years, there is no specific etiology, objective diagnostic criteria, or drug treatment. However, up-regulation of inflammation in ASD patients has been demonstrated in blood samples. Increased peripheral inflammation could have devastating effects on the developing brain. Peripheral inflammation in the blood could cross the blood-brain-barrier to stimulate microglia in the brain to produce aberrant levels of cytokines that regulate neuroinflammation such as insulin-like growth factor one (IGF1) that could alter neuronal cell-surface expression and neurotransmission. Additionally, arginase serves as a marker of inflammation, produced and expressed during cellular remodeling during brain injury. A balance of neurotransmitters, glutamate and gamma-aminobutyric acid (GABA), is critical to facilitate inter-regional signaling in the brain. Alterations of inflammatory molecules and the effects on glutamatergic neurons ability to uptake GABA in certain brain areas is currently unknown in ASD. Pathological changes in brain areas associated with social behaviors have been identified in postmortem tissue from ASD donors when compared to typically developing (TD) age and gender matched control tissue, as well as, in imaging scans of living individuals with ASD. We hypothesize that expression of inflammatory related molecules are increased in the identified brain areas related to symptoms of ASD and can be associated with altered gene expression changes in neurons as shown by gamma-aminobutyric acid type A receptor alpha 1 subunit (GABRA1). Dysfunction of GABRA1 on glutamatergic neurons could disrupt the typical neuronal balance of glutamate and GABA signaling. Inflammatory markers, IGF1 and insulin-like growth factor one receptor (IGF1R), were evaluated using quantitative polymerase chain reaction (QPCR). Additionally, IGF1 and arginase were evaluated using immunohistochemistry in both white and gray matter from the anterior cingulate cortex (ACC). Laser capture microdissection (LCM) was used to obtain single cell captures of glutamatergic neurons. IGF1R and GABRA1 gene expression was measured using end point PCR. A significant increase in IGF1 expression was obtained in the white matter punch in comparison to typically developed age-matched subjects using QPCR during initial statistical significance, however, was ultimately not significant. Additionally, IGF1R expression was significantly increased in ASD neurons in comparison to TD subjects utilizing the LCM method. However, a decrease expression in GABRA1 trended significance indicating a possible alteration in the neuron’s ability to facilitate proper signaling. These findings are the foundation of future investigations of signaling pathways in ASD that may uncover cell-specific etiologies and drug therapies for a condition that is only projected to increase in prevalence.

Autism spectrum disorder (ASD) is a neurodevelopmental disability affecting communication and social behaviors. Research is needed because the percentage of children affected by ASD is 1 in 59, and it is diagnosed in males at a rate of 1 in 42. Animal models must be used, because the neurological changes that lead to ASD occur during prenatal development. In this study, three mouse models were used to represent possible causes of ASD. The BTBR model is a genetically engineered model that displays social behavior deficits and has neuroanatomical findings similar to ASD. The other models include the Poly-IC and valproic acid injected mice which exposes the pregnant mother to a virus activating her immune system or a drug thought to affect brain development, respectively. In all three models the effects of brain-derived neurotrophic factor or BDNF, which is an important cytokine in the brain responsible for synaptic plasticity, maintenance and recognition, are being studied via expression levels of NTRK2. BDNF activates cell signaling cascades in glutamatergic neurons via the TrkB receptor which is encoded by the NTRK2 gene. It was previously found that NTRK2 expression was reduced in glutamatergic cells in people affected by ASD. The first outcome of the study is to determine gene expression differences in glutamatergic neurons captured from the cingulate cortex in all three models as well as in wild type control mice. Additionally, a second outcome of the study is to optimize a new protocol for single cell gene expression using a nested PCR method. This was done by comparing the previously used method for relative end-point PCR with the nested method to identify gene expression alterations. To prepare for the two PCR methods, samples were dehydrated and laser capture microdissection was performed on mouse brain tissue to obtain pyramidal neurons from the cingulate area. This area is highly connected to the limbic system and plays a role in personality and communication. All animal procedures were approved by the ETSU animal care committee. RNA isolation was performed on 1000 cells after which RNA was reverse transcribed into cDNA using the Superscript III cDNA synthesis system. Initial optimization experiments included using various amounts of starting cDNA and determining expression differences using relative end-point PCR and Agilent tape station. The same starting cDNA was used and initially 20 cycles of PCR were performed using Prime5 HotStart Master Mix followed by a quantitative PCR reaction using Powerup on the BioRad CFX96 RT detection system. Gene expression was performed using NTRK2 as the target gene and GAPDH as the reference gene for each method. Both methods will allow the detection of changes in the expression levels of NTRK2 and GAPDH when different sample concentrations are used. This data could help establish a link between maternal immune system activation or exposure to certain drugs during pregnancy with the occurrence of ASD.

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Cerebrovascular accidents are a leading cause of morbidity and mortality in the United States. Many conditions exist which may mimic this disease process including seizures, migraines, metabolic derangements, infections, space-occupying lesions, neurodegenerative disorders, peripheral neuropathy, cervical myelopathy, syncope, other vascular disorders, and functional neurologic disorder. Timely diagnosis and treatment are important in order to preserve functional status in these patients. A 48-year-old male presented to the emergency department with a 28-hour history of worsening left sided numbness, tingling, weakness, and feeling off balance. The patient stated that for the past several months he had noticed these symptoms, but they suddenly became worse the day prior. He also described shooting pains down the left arm with certain movements of his neck. The patient denied any difficulty with speaking, understanding words, performing mental tasks, bowel or bladder incontinence, or right sided symptoms. Physical exam showed intact cranial nerves II-XII, 5/5 strength of upper and lower extremities on the right side, 4/5 strength of upper and lower extremities on the left side. Romberg test was normal, heel to shin and finger to nose were intact bilaterally. Foot drop was noted on the left side and placement of the foot on the ground was noted to be clumsy. Initial head CT in the emergency department showed a frontal lobe hypodensity and was without intracranial hemorrhage. Computed tomography angiography of the head and neck showed no large vessel thrombosis or stenosis. Echocardiography revealed normal chamber sizes, normal left ventricular ejection fraction, no patent foramen ovale, and no left atrial or left ventricular thrombus. Telemetry monitoring throughout the stay remained sinus rhythm. Magnetic resonance imaging of brain and cervical spine was performed showing multifocal acute infarcts of the right and left frontal lobes and severe cervical spondylosis at C4-C6 with spinal cord edema in T2 sequences slightly below that level. The patient subsequently underwent a cervical spine decompression for the spinal cord compression during the hospital stay. Due to the multifocal lesions noted on the brain MRI, a vasculitis workup was performed which returned negative for any abnormal test findings. The patient was also diagnosed with diabetes mellitus type 2 during the stay as he was found to have a glycosylated hemoglobin A1C of >12. He was initially hypertensive during hospitalization, but this resolved on its own after day three of the hospitalization so anti-hypertensives were not required. The patient was discharged home on high intensity statin therapy, dual oral hypoglycemic therapy for his diabetes mellitus, home physical therapy, and he was scheduled to start dual antiplatelet therapy seven days after cervical spine surgery. This dual antiplatelet therapy with clopidogrel and aspirin was to be continued for three weeks after which continuation with low dose aspirin was advised. As seen in this case, patients that present with a cerebrovascular accident should always be evaluated for other etiology behind his or her symptoms and having a low threshold for pursing other additional diagnoses is reasonable.

Title: Hypersensitive and circadian effects of acebutolol administration in Scn1b-/- mice. Rationale: Dravet syndrome (DS) is a severe form of pediatric epilepsy with characterizations of pharmacoresistant seizures and developmental delay. A rarer variant of the DS model is caused by homozygous loss-of-function mutations in SCN1B, which is essential in regulating sodium channel gating, expression, localization, and the firing of action potentials. Mutations in SCN1B result in severe seizures as well as a higher risk of Sudden Unexpected Death in EPilepsy (SUDEP). Factors underlying SUDEP are poorly understood, although cardiac arrhythmias have been implicated. Acebutolol (ACE) is a common beta-blocker used in the treatment of arrhythmias and hypertension. We hypothesized that treating mice with ACE will decrease cardiac arrhythmias and the incidence of SUDEP, prolonging lifespan of Scn1b null mice. Methods: Wild-type (WT) and null (KO) mice were given daily injections of 10 mg/kg ACE or saline starting at postnatal day 15 (after typical seizure onset) either during the day (09:00) or at night (21:00). In the day group, ECG was recorded daily from P13 until animal death. Starting at P15 mice were recorded both pre- and post- injection to analyze the long-term and acute effects of treatment. Results: A modest, but significant, increase in survival curves in our KO animals was observed compared to saline treated mice for those given injections during the day (a 2 day increase in median survival). In addition, in this group, the onset of animal death was delayed. To investigate the timing of drug delivery, a subset of mice was given injections at night. In this group there was actually a decrease in lifespan, with an earlier onset of death compared to saline treated mice. On a daily basis from P13, the heart rate (HR) of KO mice was significantly lower than WT but remained steady until the day prior to animal death. HR the day prior to death consistently dropped ~50% (average 414 bpm to 193 bpm) in our saline group; this was prevented in KO animals treated with ACE (421 bpm). Analysis of acute recordings following ACE administration showed that KO mice had a significantly larger reduction in heart rate compared to WT (38% vs. 11%). Further analysis of heart rate variability in these recordings demonstrated that RMSSD (a measure of vagal control of the heart) was reduced in KO mice, with differences in both baseline and following ACE administration. Conclusions: Leading up to death, we believe it is possible ACE assisted in decreased cardiovascular deficits that could lead to SUDEP and contributed to the modestly increased lifespan. In addition, our results demonstrate the importance of timing in delivery of drugs targeted at SUDEP. Finally, these results suggest that there is a possible hypersensitivity to beta-adrenergic blockade in Scn1b-/- mice. Funding: This work was supported by a grant from the Research Development Committee at East Tennessee State University and NIH grant R21NS116647 to C.R.F.

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