The accurate detection and quantification of human diabetic somatic polyneuropathy (DSPN) are important to define at risk patients, anticipate deterioration, and assess new therapies. Current methods lack sensitivity, require expert assessment and have major shortcomings when employed to define therapeutic efficacy. In recent years, in vivo corneal confocal microscopy (IVCCM) has shown potential as a surrogate endpoint for DSPN.This study aims to investigate fundamental aspects of IVCCM such as repeatability and optimal scanning methodology and establish changes in corneal nerve morphology in relation to the severity of DSPN and regeneration in response to normalisation of hyperglycaemia. Furthermore, it aims to provide a novel fully automated image analysis algorithm for the quantification of corneal nerve morphology and establish the diagnostic ability of CCM.IVCCM shows high repeatability which is enhanced with more experienced observers. Central corneal innervation is comparable to adjacent peripheral innervation in mild diabetic neuropathy but the central cornea may be more sensitive to change. Corneal nerve loss is symmetrical and progressive with increasing neuropathic severity and corneal nerves show significant regenerative capacity following rapid normalisation of glycaemic control after simultaneous pancreas and kidney transplantation. The novel image analysis algorithm strongly correlates with human expert annotation and therefore represents a rapid, objective and repeatable means of assessing corneal nerve morphology. Automated image quantification may replace human manual assessment with high diagnostic validity for DSPN.

Neuropathy is one of the main long term complications of diabetes affecting 30-50% of patients. It is the major contributing factor for foot ulceration with a life time risk which may be as high as 25%. Hence neuropathy leads to reduced pain and pressure perception, anatomic deformities and an impaired microcirculation. More specifically, unperceived minor trauma results in cutaneous injury which when combined with an inadequate pressure induced vasodilator response leads to tissue breakdown and ulceration. Once ulcers form, healing may be delayed or difficult to achieve, particularly if infection occurs in the deeper tissues and bone which can then lead to amputation. In the UK, South Asians (people originating from India, Pakistan and Bangladesh) have an excess mortality for coronary artery disease (CAD), stroke and end-stage renal disease when compared to white Europeans. However, it has been shown that South Asian people with type 2 diabetes in the UK are only one third as likely to have a foot ulcer compared with White European diabetic patients. This has been attributed to lower levels of peripheral neuropathy in Asians, but has not been systematically explored in detail. In the present study, both neurological and vascular deficits in a group of South Asian and European patients with type II diabetes have been assessed. The results demonstrate that: • South Asian diabetic patients have poorer glycaemic control, but paradoxically lower triglycerides. This finding may be relevant to the finding that they have a lower incidence of neuropathy, as triglycerides have been related to neuropathy and foot ulceration. • South Asians compared to Europeans have better small fibre function and a trend for better structure (Intra epidermal nerve fibre density and corneal nerve morphology) and large fibre function assessed with nerve conduction studies. • South Asians have higher foot skin oxygenation and hyperaemic blood flow response to heating. • South Asians have a thicker epidermis and a trend for a better capillary density. Therefore these alterations may protect South Asians from the development of foot ulceration.

Hereditary peripheral neuropathy, known asCharcot Marie Tooth disease (CMT) and with an incidence of 1:2500 -1:10 000, is the most common hereditary neuromuscular disorder. Type CMT 1A is the most common form of CMT refering to the group of primary demyelinitateing motor and sensory peripheral neuropathies. CMT phenotype is clinically characterized by chronic slowly progressive distal muscle weakness and atrophy with hypo or areflexia and mild to moderate acral sensory loss. The lower limbs are predominantly affected. The aims of this study were to describe the first and most common signs of CMT1A during the first decade of life, to characterize their progression, and evaluate the sensitivity of CMTNS (Charcot-Marie- Tooth neuropath scale) for CMT1A young children. Sixteen children aged 3 to 10 years with genetically proven CMT 1A were examined. All patients were clinically examined, underwent electrophysiological examination, and were scored by CMTNS. Eight were followed for up to two years. Our data shows that CMT 1A in children under the age of 10 years causes only a mild disability. Initial signs of CMT 1A were difficulty in heel walking (15/16, 93%) and lower limb hypo or areflexia ( 13/16, 81%). The test of heel walking can be easily used as a screening test for hereditary neuropathies in pediatrics....

Great strides have been made with regard to neurogenesis in the enteric nervous system (ENS), rapidly following in the wake of recent revelations about the neurogenic properties of the central nervous system (CNS). As the ENS is more exposed, highly complex, and vulnerable to a variety of developmental, acquired, and multisystemic disorders, there is a sense of urgency for studies to address the potential within the gut to restore neurons that are injured or lost. It is the intricacies of the ENS and yet unclear relationships between agonists and embryonic precursors that have made demonstrating the arrival of new neurons in mature gut difficult under steady-state conditions. This thesis demonstrates that inducible models of a wide range of insults to the gut have yielded crucial information about ENS neurogenesis, while in vivo experimentation under steady-state conditions has proven inconsistent. Specifically, the signaling pathways of Ret and PTEN have revealed the existence of a ‘natural block’ that normally regulates neurogenesis and keeps proliferation well controlled. Furthermore, the overwhelming effects of serotonin agonism on neuron density in response to injury have uncovered an essential role of neuronal transdifferentiation by enteric glial cells that extends beyond what was once understood to be a largely homeostatic role. The influence of extrinsic innervation of the gut will also be explored, physiology of which is important for both the utility of gut microbiota and the role of Schwann cell progenitors in the development of the ENS. Therefore, this thesis will outline ENS organization and function, as well as describe common pathologies that serve as the foundation upon which neurogenesis is investigated. Critical inducible models to which chemical and molecular agonists are applied will be examined in detail, as it is through these models that therapeutics and biomedical engineering can be optimized in order to correct the pathophysiology of enteric neuropathies that as of now are still treated with surgical intervention.

To investigate the effects of chronic diabetes on baroreflex control of renal sympathetic nerve activity (RSNA), OVE26 diabetic (transgenic mouse line which develops hyperglycemia within the first 3 weeks after birth) and FVB control mice 5-6 months old were studied. Under anesthesia, RSNA in response to sodium nitroprusside (SNP)- and phenylephrine (PE)-induced mean arterial pressure changes (ΔMAP) were measured. Baroreflex-induced inhibition of RSNA during PE infusion was characterized using the sigmoid logistic function curve. Baroreflex-induced excitation of RSNA during SNP infusion was characterized by the RSNA vs. ΔMAP relationship. Mean arterial pressure (MAP) responses to the left aortic depressor nerve (ADN) stimulation were evaluated. Compared to FVB control, we found in OVE26 mice that (1) RSNA in response to MAP increase during PE infusion was dramatically reduced, as characterized by the maximal gain of the RSNA sigmoid logistic function curve (FVB: -20.0±5.1; OVE26: -7.6±0.8%/mm Hg, P<0.05); (2) RSNA in response to MAP decrease during SNP infusion was also attenuated (P<0.05); (3) MAP responses to ADN stimulation were reduced (P<0.05). We concluded that chronic diabetes impairs baroreflex control of RSNA in OVE26 diabetic mice. The use of the transgenic OVE26 diabetic mouse model may underlie a foundation for the further understanding of diabetes-induced autonomic neuropathy.

Baroreflex control of heart rate (HR) is impaired in diabetes mellitus. We hypothesized that diabetes mellitus induced functional changes of neural components at multiple sites within the baroreflex arc. Type 1 diabetic mice (OVE26) and FVB control mice were anesthetized. Baroreflex-mediated HR responses to sodium nitroprusside- (SNP) and phenylephrine- (PE) induced mean arterial blood pressure (MAP) changes were measured. Baroreceptor function was characterized by measuring the percent (%) change of baseline integrated aortic depressor nerve activity (Int ADNA) in response to SNP- and PE-induced MAP changes. The HR responses to electrical stimulation of the left aortic depressor nerve (ADN) and the right vagus nerve were assessed. Compared with FVB control mice, we found in OVE26 mice that (1) baroreflex-mediated bradycardia and tachycardia were significantly reduced. (2) The baroreceptor afferent function in response to MAP increase did not differ, as assessed by the parameters of the logistic function curve. But, the inhibition of Int ADNA in response to MAP decrease was significantly attenuated. (3) The maximum amplitude of bradycardic responses to right vagal efferent stimulation was augmented. (4) In contrast, the maximum amplitude of bradycardic responses to left ADN stimulation was decreased. Since Int ADNA was preserved in response to MAP increase and HR responses to vagal efferent stimulation were augmented, we conclude that a deficit of the central mediation of baroreflex HR contributes to the overall attenuation of baroreflex sensitivity in OVE26 mice. The successful conduction of physiological experiments on the ADN in OVE26 mice may provide a foundation for the understanding of cellular and molecular mechanisms of diabetes-induced cardiac neuropathy.

BACKGROUND: Containing a brain for signal processing and decision making, and a peripheral component for sensation and response, the nervous system provides higher organisms a powerful method of interacting with their environment. The specific neurons involved in pain sensation are known as nociceptors and are the source of normal nociceptive pain signaling to prompt appropriate responses. Though acute hypersensitization can be advantageous by encouraging an organism to allow an injured area to heal, chronic pain conditions can be pathological and can markedly reduce quality of life. While a variety of genes have been associated with congenital pain conditions, two rare cases examined in this study have not had their mutated genes identified. Potassium voltage-gated channel subfamily H member 8, or KCNH8, is involved in regulating action potential production and propagation, and has not been linked with pain processing of any kind to date. Here, a male patient evaluated at Boston Children’s Hospital contains a novel single-base KCNH8 mutation and possesses an extremely low sensitivity to cold temperatures and mechanical pain, but a higher sensitivity to warmer temperatures. A separate protein, intersectin-2, or ITSN2, normally functions in clathrin-mediated endocytosis and exocytosis. A second patient at Boston Children’s Hospital expresses a previously-unseen point mutation in ITSN2 and experiences erythromelalgia, characterized by episodes of intense pain and red, swollen limbs during ambient warm temperatures. Through the use of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 genome editing, this study will produce these specific genetic mutations in mouse lines to explore their effects on mammalian behavior. OBJECTIVES: This project employs two transgenic mouse models to study the behavioral phenotypes associated with rare potentially damaging mutations in KCNH8 and ITSN2 exhibited in the human patients. Through these experiments, a greater understanding of neural pain signaling and sensitivity changes can occur. METHODS: The differences in temperature preference of KCNH8 and ITSN2 mutant mice compared to wild type mice lacking these mutations was studied using thermal plates under cold and warm conditions. Direct application of acetone and von Frey filaments to mouse paws was used to study cold and mechanical sensitivity. Further testing of stamina, anxiety, coordination, and strength were also evaluated. RESULTS: A marked decrease in sensitivity to von Frey stimulation (p<0.01) and acetone administration (p<0.05) was observed in KCNH8 mutant mice. Thermal preference testing demonstrated a decreased preference for warmer temperatures as compared to wild type mice. In addition, anxiety levels were also observed to be slightly higher in these mutant KCNH8 mice (p<0.05). The mutant ITSN2 mice spent less time at cooler temperatures, though surprisingly they significantly preferred warmer conditions as compared to their wild type littermates. A full and partial reversal of these temperature preferences was demonstrated in cold and heat thermal conditions respectively after intraperitoneal gabapentin injection, which normalized the mice toward wild type behavior. CONCLUSIONS: Data from the KCNH8 mutant mouse model indicates an aversion to warmer temperatures and a decreased ability to detect cold or mechanical pressure, much like the human patient. The mutant ITSN2 mice were less likely to spend time at cooler temperatures, indicating heightened sensory sensitivity, but their preference for warmer temperatures suggests a possible desensitization of the affected nociceptors. These results often mirror the patient’s phenotype, but the preference for ambient warmer environments appears opposite to the patient. As the ITSN2 mice feel discomfort at cooler temperatures, a proposed desensitization at warmer temperatures would result in a more comfortable environment and could explain the observed preference. The trends toward normal neural firing rates achieved through gabapentin injection suggest that the aberrant responses in mutant ITSN2 mice is due to altered sensitization, but additional examination under these conditions with a larger group of mice is necessary to further unravel these signaling pathways. However, these extremely encouraging data introduce two new molecular targets for acute pain control.

Indiana University-Purdue University Indianapolis (IUPUI) / My first aim identified enrollment patterns and variables that predict enrollment in a diverse underserved population and evaluated barriers to enrollment. We analyzed data from the INGENIOUS, (Indiana GENomics Implementation and Opportunity for the UnderServed), pharmacogenomics implementation clinical trial conducted at a community hospital for underserved subjects and a statewide healthcare system. Our main finding revealed, African-Americans were less likely to refuse the study than non-Hispanic Whites (Safety net, OR =0.68, p<0.002; Academic hospital, OR=0.64, p<0.001), using a logistic regression model. The most frequent barriers to enrollment included not being interested, being too busy, transportation, and illness in African-American and non-Hispanic White subjects. In conclusion, improving research awareness, widening the inclusion criteria, and hiring recruiters who represent potential enrollees, should improve enrollment in African-Americans and other diverse populations. My 2nd research aim evaluated racial/ethnic differences in pharmacokinetics, safety, efficacy, and pharmacogenetics in 213 new molecular entities (NMEs). The current approved drug label for NMEs between 2014 to 2018 was updated in the FDA database. A qualitative analysis revealed ~ 9% (n=20/213) of NMEs reported racial/ethnic differences in the approved product label for PK, safety, efficacy, and/or pharmacogenetics. In conclusion, evaluating racial/ethnic differences in drug exposure and response early in the drug development program is essential to providing recommendations for different racial/ethnic subpopulations. My final aim 3, identified genetic biomarkers of Taxane Induced Peripheral Neuropathy (TIPN) in African-American breast cancer patients. We used an innovative computational tool, ALDY, to identify genetic variants in CYP2C8, CYP3A4, and CYP3A5 in 207 breast cancer subjects. TaqMan SNP genotyping for SNP, rs776746 (T>C) was performed in 160 subjects. Subjects were collapsed into three metabolizer groups; normal, intermediate, and poor metabolizer to test the association of peripheral neuropathy, dose reductions and CYP2C8/CYP3A5 metabolizer status. A logistic regression revealed CYP2C8 metabolizer status is associated with grades 3-4 peripheral neuropathy (p=0.04, OR= 2.21). CYP2C8*2 was modestly associated with dose reductions. In conclusion, evaluating pharmacogenetic and pharmacokinetic studies of paclitaxel and CYP2C8 is important. These studies may lead to clinical actionable prescribing of paclitaxel and improve the tolerance and efficacy in African-American breast cancer patients. / 2022-02-16

No description available.

Despite the development of antiretroviral therapy (ART), human immunodeficiency virus (HIV)-associated distal sensory polyneuropathy (HIV-DSP) has remained prevalent. To recapitulate chronic pain during HIV infection, simian immunodeficiency virus (SIV)-infected rhesus macaques were examined to dissect the molecular mechanisms of peripheral and central sensitization. Previous studies identified atrophy in nociceptive neurons during SIV infection, which was associated with monocyte infiltration into the dorsal root ganglia (DRG). However, the sensory signaling mechanism connecting this pathology to symptomology remains unclear, especially because pain persists after resolution of high viremia and inflammation with ART. We hypothesize that residual DRG and dorsal horn neuroinflammation contributes to nociceptive sensitization. Using three cohorts of macaques (uninfected, SIV-infected, and SIV+/ART), we showed an increase in the cellular and cytokine inflammatory profiles in the DRG of SIV+/ART macaques compared to uninfected animals. We observed significant increased expression of nociceptive ion channels, transient receptor potential vanilloid 1 (TRPV1) and ankyrin 1 (TRPA1) among DRG neurons in SIV+/ART compared to uninfected animals. SIV-infected and SIV+/ART animals showed reduced innervation of the non-peptidergic nociceptors into the dorsal horn compared to uninfected animals. Finally, there were significantly higher numbers of CD68+ macrophages in the dorsal horn of SIV+/ART macaques compared to uninfected animals. As pain persists in people with HIV (PWH) on ART, mechanisms outside of viral load and DRG pathology were investigated. Here, bulk RNAseq of DRGs from the three cohorts of macaques revealed a significant increase in upstream regulation by interferon γ (IFNγ) and lipopolysaccharide (LPS) with SIV infection. The introduction of ART decreased the signaling of IFNγ and LPS, yet in SIV+/ART animals, there was a significant increase in expression of the IFNγ inducible chemokines, CXCL9 and CXCL10. To assess if CXCL9 and/or CXCL10 influence TRPV1/TRPA1 expression, we utilized an induced pluripotent stem cell-derived peripheral sensory neuron model (IPSC-PSN). Treatment of IPSC-PSNs with CXCL9 and/or CXCL10 induced a significant increase in TRPV1, but not TRPA1 expression. Since macrophages persist in the DRGs of SIV+/ART macaques, monocyte-derived macrophages (MDMs) were treated with IFNγ or LPS. MDMs treated with IFNᵧ but not LPS, significantly increased the release of CXCL9/CXCL10. Conditioned media from MDMs treated with IFNγ, but not LPS, significantly increased expression of TRPV1 in IPSC-PSNs. Inhibition of the CXCL9/CXCL10 receptor, CXCR3, in IPSC-PSNs partially blocked TRPV1 upregulation following treatment with conditioned media from IFNᵧ treated MDMs. In summary, these data demonstrate that neuroinflammation, characteristics of nociceptor sensitization, and central terminal atrophy persists in SIV infection with ART. Mechanistically, these data indicate a potential role of IFNᵧ signaling in the sensitization of nociceptors in HIV-DSP despite ART. / Biomedical Sciences