Tumor-liver interface in MRI of liver metastasis enables prediction of EGFR mutation in patients with lung cancer: A proof-of-concept study.

BACKGROUND: Preoperative prediction of the epidermal growth factor receptor (EGFR) status in non-small-cell lung cancer (NSCLC) patients with liver metastasis (LM) may have potential clinical values for assisting in treatment decision-making. PURPOSE: To explore the value of tumor-liver interface (TLI)-based magnetic resonance imaging (MRI) radiomics for detecting the EGFR mutation in NSCLC patients with LM. METHODS: This retrospective study included 123 and 44 patients from hospital 1 (between Feb. 2018 and Dec. 2021) and hospital 2 (between Nov. 2015 and Aug. 2022), respectively. The patients received contrast-enhanced T1-weighted (CET1) and T2-weighted (T2W) liver MRI scans before treatment. Radiomics features were extracted from MRI images of TLI and the whole tumor region, separately. The least absolute shrinkage and selection operator (LASSO) regression was used to screen the features and establish radiomics signatures (RSs) based on TLI (RS-TLI) and the whole tumor (RS-W). The RSs were evaluated by the receiver operating characteristic (ROC) curve analysis. RESULTS: A total of 5 and 6 features were identified highly correlated with the EGFR mutation status from TLI and the whole tumor, respectively. The RS-TLI showed better prediction performance than RS-W in the training (AUCs, RS-TLI vs. RS-W, 0.842 vs. 0.797), internal validation (AUCs, RS-TLI vs. RS-W, 0.771 vs. 0.676) and external validation (AUCs, RS-TLI vs. RS-W, 0.733 vs. 0.679) cohort. CONCLUSION: Our study demonstrated that TLI-based radiomics can improve prediction performance of the EGFR mutation in lung cancer patients with LM. The established multi-parametric MRI radiomics models may be used as new markers that can potentially assist in personalized treatment planning.

Autonomic Dysreflexia in Spinal Cord Injury: Mechanisms and Prospective Therapeutic Targets.

High-level spinal cord injury (SCI) often results in cardiovascular dysfunction, especially the development of autonomic dysreflexia. This disorder, characterized as an episode of hypertension accompanied by bradycardia in response to visceral or somatic stimuli, causes substantial discomfort and potentially life-threatening symptoms. The neural mechanisms underlying this dysautonomia include a loss of supraspinal control to spinal sympathetic neurons, maladaptive plasticity of sensory inputs and propriospinal interneurons, and excessive discharge of sympathetic preganglionic neurons. While neural control of cardiovascular function is largely disrupted after SCI, the renin-angiotensin system (RAS), which mediates blood pressure through hormonal mechanisms, is up-regulated after injury. Whether the RAS engages in autonomic dysreflexia, however, is still controversial. Regarding therapeutics, transplantation of embryonic presympathetic neurons, collected from the brainstem or more specific raphe regions, into the injured spinal cord may reestablish supraspinal regulation of sympathetic activity for cardiovascular improvement. This treatment reduces the occurrence of spontaneous autonomic dysreflexia and the severity of artificially triggered dysreflexic responses in rodent SCI models. Though transplanting early-stage neurons improves neural regulation of blood pressure, hormonal regulation remains high and baroreflex dysfunction persists. Therefore, cell transplantation combined with selected RAS inhibition may enhance neuroendocrine homeostasis for cardiovascular recovery after SCI.

Sprouting of afferent and efferent inputs to pelvic organs after spinal cord injury.

Neural plasticity occurs within the central and peripheral nervous systems after spinal cord injury (SCI). Although central alterations have extensively been studied, it is largely unknown whether afferent and efferent fibers in pelvic viscera undergo similar morphological changes. Using a rat spinal cord transection model, we conducted immunohistochemistry to investigate afferent and efferent innervations to the kidney, colon, and bladder. Approximately 3-4 weeks after injury, immunostaining demonstrated that tyrosine hydroxylase (TH)-labeled postganglionic sympathetic fibers and calcitonin gene-related peptide (CGRP)-immunoreactive sensory terminals sprout in the renal pelvis and colon. Morphologically, sprouted afferent or efferent projections showed a disorganized structure. In the bladder, however, denser CGRP-positive primary sensory fibers emerged in rats with SCI, whereas TH-positive sympathetic efferent fibers did not change. Numerous CGRP-positive afferents were observed in the muscle layer and the lamina propria of the bladder following SCI. TH-positive efferent inputs displayed hypertrophy with large diameters, but their innervation patterns were sustained. Collectively, afferent or efferent inputs sprout widely in the pelvic organs after SCI, which may be one of the morphological bases underlying functional adaptation or maladaptation.

Effect of GR205171 on autonomic dysreflexia induced by colorectal distension in spinal cord injured rats.

STUDY DESIGN: Preclinical pharmacology. OBJECTIVES: To determine whether blocking substance P signaling attenuates the hypertension and bradycardia evoked by colorectal distension (CRD) in spinal cord injured (SCI) rats. SETTING: University laboratory in Pennsylvania, U.S.A. METHODS: Tachykinin NK1 receptor antagonists were administered 30 min prior to CRD three weeks after complete spinal cord transection at the 4th thoracic (T4) level. The dose range, route of administration, and pretreatment time was based on published data demonstrating occupancy of brain NK1 receptors in rodents. RESULTS: Subcutaneous (SC) administration of 10-30 mg/kg GR205171 ((2S,3S)-N-[[2-methoxy-5-[5-(trifluoromethyl)tetrazol-1-yl]phenyl]methyl]-2-phenylpiperidin-3-amine dihydrochloride) reduced CRD-induced hypertension and bradycardia by 55 and 49%, respectively, compared with pretreatment values. There was no effect of GR205171 on resting blood pressure or heart rate. In contrast, the same dose range of CP-99,994 ((2S,3S)-N-[(2-methoxyphenyl)methyl]-2-phenyl-3-piperidinamine dihydrochloride) had no effect on CRD-induced cardiovascular responses. CONCLUSIONS: The effective dose range of GR205171 to alleviate autonomic dysreflexia is consistent with the blockade of NK1 receptors on pelvic sensory afferents in the lumbosacral spinal cord, which may in turn prevent the over-excitation of sympathetic preganglionic neurons (SPNs) that regulate blood pressure and heart rate. The findings provide preclinical support for the utility of NK1 receptor antagonists to treat autonomic dysreflexia in people with SCI. The difference in the effects of the two NK1 receptor antagonists may reflect the ~200-fold lower affinity of CP-99,994 than GR205171 for the rat NK1 receptor.

Brain-Tumor Interface-Based MRI Radiomics Models to Determine EGFR Mutation, Response to EGFR-TKI and T790M Resistance Mutation in Non-Small Cell Lung Carcinoma Brain Metastasis.

BACKGROUND: Preoperative assessment of epidermal growth factor receptor (EGFR) status, response to EGFR-tyrosine kinase inhibitors (TKI) and development of T790M mutation in non-small cell lung carcinoma (NSCLC) patients with brain metastases (BM) is important for clinical decision-making, while previous studies were only based on the whole BM. PURPOSE: To investigate values of brain-to-tumor interface (BTI) for determining the EGFR mutation, response to EGFR-TKI and T790M mutation. STUDY TYPE: Retrospective. POPULATION: Two hundred thirty patients from Hospital 1 (primary cohort) and 80 patients from Hospital 2 (external validation cohort) with BM and histological diagnosis of primary NSCLC, and with known EGFR status (biopsy) and T790M mutation status (gene sequencing). FIELD STRENGTH/SEQUENCE: Contrast-enhanced T1-weighted (T1CE) and T2-weighted (T2W) fast spin echo sequences at 3.0T MRI. ASSESSMENT: Treatment response to EGFR-TKI therapy was determined by the Response Evaluation Criteria in Solid Tumors. Radiomics features were extracted from the 4 mm thickness BTI and selected by least shrinkage and selection operator regression. The selected BTI features and volume of peritumoral edema (VPE) were combined to construct models using logistic regression. STATISTICAL TESTS: The performance of each radiomics model was evaluated using the area under the receiver operating characteristic (ROC) curve (AUC). RESULTS: A total of 7, 3, and 3 features were strongly associated with the EGFR mutation status, response to EGFR-TKI and T790M mutation status, respectively. The developed models combining BTI features and VPE can improve the performance than those based on BTI features alone, generating AUCs of 0.814, 0.730, and 0.774 for determining the EGFR mutation, response to EGFR-TKI and T790M mutation, respectively, in the external validation cohort. DATA CONCLUSION: BTI features and VPE were associated with the EGFR mutation status, response to EGFR-TKI and T790M mutation status in NSCLC patients with BM. EVIDENCE LEVEL: 3 Technical Efficacy: Stage 2.

Radiomics for Detection of the EGFR Mutation in Liver Metastatic NSCLC.

RATIONALE AND OBJECTIVES: The research aims to investigate whether MRI radiomics on hepatic metastasis from primary nonsmall cell lung cancer (NSCLC) can be used to differentiate patients with epidermal growth factor receptor (EGFR) mutations from those with EGFR wild-type, and develop a prediction model based on combination of primary tumor and the metastasis. MATERIALS AND METHODS: A total of 130 patients were enrolled between Aug. 2017 and Dec. 2021, all pathologically confirmed harboring hepatic metastasis from primary NSCLC. The pyradiomics was used to extract radiomics features from intra- and peritumoral areas of both primary tumor and metastasis. The least absolute shrinkage and selection operator (LASSO) regression was applied to identify most predictive features and to develop radiomics signatures (RSs) for prediction of the EGFR mutation status. The receiver operating characteristic (ROC) curve analysis was performed to assess the prediction capability of the developed RSs. RESULTS: A RS-Primary and a RS-Metastasis were derived from the primary tumor and metastasis, respectively. The RS-Combine by combination of the primary tumor and metastasis achieved the highest prediction performance in the training (AUCs, RS-Primary vs. RS-Metastasis vs. RS-Combine, 0.826 vs. 0.821 vs. 0.908) and testing (AUCs, RS-Primary vs. RS-Metastasis vs. RS-Combine, 0.760 vs. 0.791 vs. 0.884) set. The smoking status showed significant difference between EGFR mutant and wild-type groups (p < 0.05) in the training set. CONCLUSION: The study indicates that hepatic metastasis-based radiomics can be used to detect the EGFR mutation. The developed multiorgan combined radiomics signature may be helpful to guide individual treatment strategies for patients with metastatic NSCLC.

Preoperative MRI-Based Radiomics of Brain Metastasis to Assess T790M Resistance Mutation After EGFR-TKI Treatment in NSCLC.

BACKGROUND: Preoperative assessment of the acquired resistance T790M mutation in patients with metastatic non-small cell lung cancer (NSCLC) based on brain metastasis (BM) is important for early treatment decisions. PURPOSE: To investigate preoperative magnetic resonance imaging (MRI)-based radiomics for assessing T790M resistance mutation after epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) treatment in NSCLC patients with BM. STUDY TYPE: Retrospective. POPULATION: One hundred and ten primary NSCLC patients with pathologically confirmed BM and T790M mutation status assessment from two centers divided into primary training (N = 53), internal validation (N = 27), and external validation (N = 30) sets. FIELD STRENGTH/SEQUENCE: Contrast-enhanced T1-weighted (T1CE) and T2-weighted (T2W) fast spin echo sequences at 3.0 T. ASSESSMENT: Forty-five (40.9%) patients were T790M-positive and 65 (59.1%) patients were T790M-negative. The tumor active area (TAA) and peritumoral edema area (POA) of BM were delineated on pre-treatment T1CE and T2W images. Radiomics signatures were built based on features selected from TAA (RS-TAA), POA (RS-POA), and their combination (RS-Com) to assess the T790M resistance mutation after EGFR-TKI treatment. STATISTICAL TESTS: Receiver operating characteristic (ROC) curves were used to assess the capabilities of the developed RSs. The area under the ROC curves (AUC), sensitivity, and specificity were generated as comparison metrics. RESULTS: We identified two features (from TAA) and three features (from POA) that are highly associated with the T790M mutation status. The developed RS-TAA, RS-POA, and RS-Com showed good performance, with AUCs of 0.807, 0.807, and 0.864 in the internal validation, and 0.783, 0.814, and 0.860 in the external validation sets, respectively. DATA CONCLUSION: Pretreatment brain MRI of NSCLC patients with BM might effectively detect the T790M resistance mutation, with both TAA and POA having important values. The multi-region combined radiomics signature may have potential to be a new biomarker for assessing T790M mutation. LEVEL OF EVIDENCE: 3 TECHNICAL EFFICACY: Stage 2.

The susceptibility of cardiac arrhythmias after spinal cord crush injury in rats.

High-level spinal cord injury (SCI) often interrupts supraspinal regulation of sympathetic input to the heart. Although it is known that dysregulated autonomic control increases the risk for cardiac disorders, the mechanisms mediating SCI-induced arrhythmias are poorly understood. Here, we employed a rat model of complete spinal cord crush injury at the 2nd/3rd thoracic (T2/3) level to investigate cardiac rhythm disorders resulting from SCI. Rats with T9 injury and naïve animals served as two controls. Four weeks after SCI, rats were implanted with a radio-telemetric device for electrocardiogram and blood pressure monitoring. During 24-h recordings, heart rate variability in rats with T2/3 but not T9 injury exhibited a significant reduction in the time domain, and a decrease in power at low frequency but increased power at high frequency in the frequency domain which indicates reduced sympathetic and increased parasympathetic outflow to the heart. Pharmacological blockade of the sympathetic or parasympathetic branches confirmed the imbalance of cardiac autonomic control. Activation of sympatho-vagal input during the induction of autonomic dysreflexia by colorectal distention triggered various severe arrhythmic events in T2/3 injured rats. Meanwhile, intravenous infusion of the ß1-adrenergic receptor agonist, dobutamine, caused greater incidence of arrhythmias in rats with T2/3 injury than naïve and T9 injured controls. Together, the results indicate that high-level SCI increases the susceptibility to developing cardiac arrhythmias likely owing to compromised autonomic homeostasis. The T2/3 crush model is appropriate for studying abnormal cardiac electrophysiology resulting from SCI.

Locomotor Exercise Enhances Supraspinal Control of Lower-Urinary-Tract Activity to Improve Micturition Function after Contusive Spinal-Cord Injury.

The recovery of lower-urinary-tract activity is a top priority for patients with spinal-cord injury. Historically, locomotor training improved micturition function in both patients with spinal cord injury and animal models. We explore whether training augments such as the supraspinal control of the external urethral sphincter results in enhanced coordination in detrusor-sphincter activity. We implemented a clinically relevant contusive spinal-cord injury at the 12th thoracic level in rats and administered forced wheel running exercise for 11 weeks. Awake rats then underwent bladder cystometrogram and sphincter electromyography recordings to examine the micturition reflex. Subsequently, pseudorabies-virus-encoding red fluorescent protein was injected into the sphincter to trans-synaptically trace the supraspinal innervation of Onuf's motoneurons. Training in the injury group reduced the occurrence of bladder nonvoiding contractions, decreased the voiding threshold and peak intravesical pressure, and shortened the latency of sphincter bursting during voiding, leading to enhanced voiding efficiency. Histological analysis demonstrated that the training increased the extent of spared spinal-cord tissue around the epicenter of lesions. Compared to the group of injury without exercise, training elicited denser 5-hydroxytryptamine-positive axon terminals in the vicinity of Onuf's motoneurons in the cord; more pseudorabies virus-labeled or c-fos expressing neurons were detected in the brainstem, suggesting the enhanced supraspinal control of sphincter activity. Thus, locomotor training promotes tissue sparing and axon innervation of spinal motoneurons to improve voiding function following contusive spinal-cord injury.

Transneuronal tracing to map connectivity in injured and transplanted spinal networks.

It has become widely appreciated that the spinal cord has significant neuroplastic potential, is not hard-wired, and that with traumatic injury and anatomical plasticity, the networks that we once understood now comprise a new anatomy. Harnessing advances in neuroanatomical tracing to map the neuronal networks of the intact and injured spinal cord has been crucial to elucidating this new spinal cord anatomy. Many new techniques have been developed to identify these networks using a variety of retrograde and anterograde tracers. One method of tracing that has become more widely used to map anatomical changes is transneuronal tracing. Viral tracers are being increasingly used to map spinal networks, leading to an advanced understanding of spinal circuitry and host-donor-host interactions between the injured spinal cord and neural transplants. This review will highlight advances in neuronal tracing, specifically using pseudorabies virus (PRV), and its use in the intact, injured, and transplanted spinal cord.

Deciphering Spinal Endogenous Dopaminergic Mechanisms That Modulate Micturition Reflexes in Rats with Spinal Cord Injury.

Spinal neuronal mechanisms regulate recovered involuntary micturition after spinal cord injury (SCI). It was recently discovered that dopamine (DA) is synthesized in the rat injured spinal cord and is involved in lower urinary tract (LUT) activity. To fully understand the role of spinal DAergic machinery in micturition, we examined urodynamic responses in female rats during pharmacological modulation of the DA pathway. Three to four weeks after complete thoracic SCI, the DA precursor L-DOPA administered intravenously during bladder cystometrogram (CMG) and external urethral sphincter (EUS) electromyography (EMG) reduced bladder overactivity and increased the duration of EUS bursting, leading to remarkably improved voiding efficiency. Apomorphine (APO), a non-selective DA receptor (DR) agonist, or quinpirole, a selective DR2 agonist, induced similar responses, whereas a specific DR2 antagonist remoxipride alone had only minimal effects. Meanwhile, administration of SCH 23390, a DR1 antagonist, reduced voiding efficiency by increasing tonic EUS activity and shortening the EUS bursting period. Unexpectedly, SKF 38393, a selective DR1 agonist, increased EUS tonic activity, implying a complicated role of DR1 in LUT function. In metabolic cage assays, subcutaneous administration of quinpirole decreased spontaneous voiding frequency and increased voiding volume; L-DOPA and APO were inactive possibly because of slow entry into the CNS. Collectively, tonically active DR1 in SCI rats inhibit urine storage and enhance voiding by differentially modulating EUS tonic and bursting patterns, respectively, while pharmacologic activation of DR2, which are normally silent, improves voiding by enhancing EUS bursting. Thus, enhancing DA signaling achieves better detrusor-sphincter coordination to facilitate micturition function in SCI rats.

Alterations of Dopamine-Related Transcripts in A11 Diencephalospinal Pathways after Spinal Cord Injury.

The diencephalic A11 nuclei are the primary source of spinal dopamine (DA). Neurons in this region project to all levels of the spinal cord. Traumatic spinal cord injury (SCI) often interrupts descending and ascending neuronal pathways and further elicits injury-induced neuronal plasticity. However, it is unknown how A11 neurons and projections respond to SCI-induced axotomy. Based on preliminary observation, we hypothesized that A11 DA-ergic neurons rostral to the lesion site might change their capacity to synthesize DA after SCI. Adult rats received a complete spinal cord transection at the 10th thoracic (T10) level. After 3 or 8 weeks, rostral (T5) and caudal (L1) spinal cord tissue was collected to measure mRNA levels of DA-related genes. Meanwhile, A11 neurons in the brain were explicitly isolated by laser capture microdissection, and single-cell qPCR was employed to evaluate mRNA levels in the soma. Histological analysis was conducted to assess the number of A11 DA-ergic neurons. The results showed that, compared to naïve rats, mRNA levels of tyrosine hydroxylase (TH), dopamine decarboxylase (DDC), and D2 receptors in the T5 spinal segment had a transient decrease and subsequent recovery. However, dopamine-ß-hydroxylase (DBH), D1 receptors, and DA-associated transcription factors did not change following SCI. Furthermore, axon degeneration below the lesion substantially reduced mRNA levels of TH and D2 in the L1 spinal segment. However, DDC transcript underwent only a temporary decrease. Similar mRNA levels of DA-related enzymes were detected in the A11 neuronal soma between naïve and SCI rats. In addition, immunostaining revealed that the number of A11 DA neurons did not change after SCI, indicating a sustention of capacity to synthesize DA in the neuroplasm. Thus, impaired A11 diencephalospinal pathways following SCI may transiently reduce DA production in the spinal cord rostral to the lesion but not in the brain.

Delivery of the 5-HT2A Receptor Agonist, DOI, Enhances Activity of the Sphincter Muscle during the Micturition Reflex in Rats after Spinal Cord Injury.

Traumatic spinal cord injury (SCI) interrupts spinobulbospinal micturition reflex pathways and results in urinary dysfunction. Over time, an involuntary bladder reflex is established due to the reorganization of spinal circuitry. Previous studies show that manipulation of serotonin 2A (5-HT2A) receptors affects recovered bladder function, but it remains unclear if this receptor regulates the activity of the external urethral sphincter (EUS) following SCI. To elucidate how central and peripheral serotonergic machinery acts on the lower urinary tract (LUT) system, we employed bladder cystometry and EUS electromyography recordings combined with intravenous or intrathecal pharmacological interventions of 5-HT2A receptors in female SCI rats. Three to four weeks after a T10 spinal transection, systemic and central blockage of 5-HT2A receptors with MDL only slightly influenced the micturition reflex. However, delivery of the 5-HT2A receptor agonist, DOI, increased EUS tonic activity and elicited bursting during voiding. Additionally, subcutaneous administration of DOI verified the enhancement of continence and voiding capability during spontaneous micturition in metabolic cage assays. Although spinal 5HT2A receptors may not be actively involved in the recovered micturition reflex, stimulating this receptor subtype enhances EUS function and the synergistic activity between the detrusor and sphincter to improve the micturition reflex in rats with SCI.

Dual-Pseudorabies Viral Tracing for Spinal Tyrosine Hydroxylase Interneurons Involved in Segmental Micturition Reflex Circuitry in Spinal Cord Injured Rats.

Traumatic spinal cord injury (SCI) often leads to urinary dysfunction. Although an involuntary micturition reflex can be established to elicit voiding with time, complications arise in the form of bladder hyper-reflexia and detrusor-sphincter dyssynergia that cause incontinence and inefficient expulsion of urine. To date, the neuronal mechanisms that underlie regulation of micturition after SCI are not well understood. We recently observed an increase of a population of tyrosine hydroxylase (TH)+ cells in the rat lumbosacral cord post-SCI, which contribute to the sustention of a low level of dopamine that modulates the recovered bladder reflex. To identify whether spinal TH+ cells are involved in the micturition reflex pathway post-SCI, two isoforms of the trans-synaptic retrograde tracer, pseudorabies virus encoding green fluorescent protein (GFP; PRV-152) or red fluorescent protein (RFP; PRV-614), were injected into the bladder detrusor or the external urethral sphincter (EUS), respectively, 3 weeks after a spinal cord transection at the 10th thoracic level (T10) in rats. Immunohistochemistry was performed to examine infected TH+ cells in the caudal cord at both 48 and 72 h post-injection. As a result, double-labeled TH+/GFP+ and TH+/RFP+ cells could be found in the superficial dorsal horn, parasympathetic nuclei, and dorsal gray commissure (lamina X) at both time points. More importantly, a shared population of TH+ interneurons (TH+/GFP+/RFP+) exists between bladder and EUS circuitry. These results suggest that spinal TH+ interneurons may coordinate activity of the bladder and EUS that occurs during micturition reflexes post-SCI.

Spinal Dopaminergic Mechanisms Regulating the Micturition Reflex in Male Rats with Complete Spinal Cord Injury.

Traumatic spinal cord injury (SCI) often causes micturition dysfunction. We recently discovered a low level of spinally-derived dopamine (DA) that regulates recovered bladder and sphincter reflexes in SCI female rats. Considering substantial sexual dimorphic features in the lower urinary tract, it is unknown if the DA-ergic mechanisms act in the male. Histological analysis showed a similar distribution of tyrosine hydroxylase (TH)+ neurons in the lower cord of male rats and the number increased following thoracic SCI. Subsequently, focal electrical stimulation in slices obtained from L6/S1 spinal segments of SCI rats elicited detectable DA release with fast scan cyclic voltammetry. Using bladder cystometrogram and external urethral sphincter (EUS) electromyography in SCI male rats, intravenous (i.v.) administration of SCH 23390, a D1-like receptor (DR1) antagonist, induced significantly increased tonic EUS activity and a trend of increased residual volume, whereas activation of these receptors with SKF 38393 did not influence the reflex. Meanwhile, blocking spinal D2-like receptors (DR2) with remoxipride had no effect but stimulating these receptors with quinpirole elicited EUS bursting to increase voiding volume. Further, intrathecal delivery of SCH 23390 and quinpirole resulted in similar responses to those with i.v. delivery, respectively, which indicates the central action regardless of delivery route. In addition, metabolic cage assays showed that quinpirole increased the voiding frequency and total voiding volume in spontaneous micturition. Collectively, spinal DA-ergic machinery regulates recovered micturition reflex following SCI in male rats; spinal DR1 tonically suppress tonic EUS activity to enable voiding and activation of DR2 facilitates voiding.

Transgene expression within the spinal cord of hTH-eGFP rats.

The enzyme tyrosine hydroxylase (TH) is broadly expressed in catecholaminergic neurons. In the spinal cord, four components contain TH, including A11 diencephalospinal dopaminergic (DA-ergic) pathways, intraspinal DA-related neurons, supraspinal noradrenergic projections, and afferents of TH-expressing sensory neurons. A human TH-enhanced green fluorescent protein (hTH-eGFP) transgenic rat was recently developed to tag TH+ profiles in the nervous system for visualization. Using immunostaining, we found that only A11 pathways express GFP whereas the other 3 components do not in the spinal cord. Thus, this may suggest a genetic difference among these TH+ elements even though they produce the same protein.

Grafting Embryonic Raphe Neurons Reestablishes Serotonergic Regulation of Sympathetic Activity to Improve Cardiovascular Function after Spinal Cord Injury.

Cardiovascular dysfunction often occurs after high-level spinal cord injury. Disrupting supraspinal vasomotor pathways affects basal hemodynamics and contributes to the development of autonomic dysreflexia (AD). Transplantation of early-stage neurons to the injured cord may reconstruct the descending projections to enhance cardiovascular performance. To determine the specific role of reestablishing serotonergic regulation of hemodynamics, we implanted serotonergic (5-HT+) neuron-enriched embryonic raphe nucleus-derived neural stem cells/progenitors (RN-NSCs) into a complete spinal cord transection lesion site in adult female rats. Grafting embryonic spinal cord-derived NSCs or injury alone served as 2 controls. Ten weeks after injury/grafting, histological analysis revealed well-survived grafts and partial integration with host tissues in the lesion site. Numerous graft-derived serotonergic axons topographically projected to the caudal autonomic regions. Neuronal tracing showed that host supraspinal vasomotor pathways regenerated into the graft, and 5-HT+ neurons within graft and host brainstem neurons were transsynaptically labeled by injecting pseudorabies virus (PRV-614) into the kidney, indicating reconnected serotonergic circuits regulating autonomic activity. Using an implanted telemeter to record cardiovascular parameters, grafting RN-NSCs restored resting mean arterial pressure to normal levels and remarkably alleviated naturally occurring and colorectal distension-induced AD. Subsequent pharmacological blockade of 5-HT2A receptors with ketanserin in RN-NSC-grafted rats reduced resting mean arterial pressure and increased heart rate in all but 2 controls. Furthermore, spinal cord retransection below RN-NSC grafts partially eliminated the recovery in AD. Collectively, these data indicate that RN-NSCs grafted into a spinal cord injury site relay supraspinal control of serotonergic regulation for sympathetic activity to improve cardiovascular function.SIGNIFICANCE STATEMENT Disruption of supraspinal vasomotor pathways results in cardiovascular dysfunction following high-level spinal cord injury. To reestablish the descending regulation of autonomic function, we transplanted serotonergic neuron enriched embryonic raphe nucleus-derived neural stem cells/progenitors into the lesion site of completely transected rat spinal cord. Consequently, grafted raphe nucleus-derived neural stem cells/progenitors acted as a neuronal relay to reconnect supraspinal center and spinal sympathetic neurons below the injury. The reconstituted serotonergic regulation of sympathetic activity led to the improvement of hemodynamic parameters and mitigated autonomic dysreflexia. Based on morphological and physiological results, this study validates the effectiveness of transplanting early-stage serotonergic neurons into the spinal cord for cardiovascular functional recovery after spinal cord injury.

Development of Cardiovascular Dysfunction in a Rat Spinal Cord Crush Model and Responses to Serotonergic Interventions.

Selection of a proper spinal cord injury (SCI) rat model to study therapeutic effects of cell transplantation is imperative for research in cardiovascular functional recovery, due to the local harsh milieu inhibiting cell growth. We recently found that a crushed spinal cord lesion can minimize fibrotic scarring and grafted cell death compared with open-dura injuries. To determine if this SCI model is applicable for studying cardiovascular recovery, we examined hemodynamic consequences following crushed SCI and tested cardiovascular responses to serotonin (5-HT) or dopamine (DA) receptor agonists. Using a radio-telemetric system, multiple cardiovascular parameters were recorded prior to, 2, and 4 weeks after SCI, including resting mean arterial pressure (MAP) and heart rate (HR), as well as spontaneous or colorectal distension (CRD)-induced autonomic dysreflexia (AD). The results showed that this injury caused tachycardia at rest as well as the occurrence of spontaneous or artificially induced dysreflexic events. Four weeks post-injury, specific activation of 5-HT2A receptors by subcutaneous (s.c.) or intrathecal (i.t.) delivery of Dimethoxy-4-iodoamphetamine (DOI) remarkably increased resting MAP levels in a dose-dependent fashion. During CRD-induced autonomic dysreflexia, systemic administration of DOI alleviated the severity of bradycardia responsive to episodic hypertension. In contrast, selective stimulation of 5-HT1A receptors with 8-OH-DPAT or non-selective activation of DA receptors with apomorphine did not affect cardiovascular performance. Thus, crush injuries induce cardiovascular abnormalities in rats that are sensitive to 5-HT2A receptor stimulation, indicating a reliable SCI model to study how cell-based approaches impact the severity of autonomic dysreflexia and identify a possible target for pharmacological interventions.

Transsynaptic tracing to dissect supraspinal serotonergic input regulating the bladder reflex in rats.

AIMS: This study was designed to determine specific cell groups of the raphe nuclei (RN) that give rise to supraspinal serotonergic projections regulating the bladder reflex. METHODS: Anesthetized rats underwent surgery to open the abdomen and expose the bladder. A total of 6 µL transsynaptic neuronal tracer pseudorabies virus (PRV-152), encoding for green fluorescent protein (GFP), was injected into the bladder detrusor. After 72 or 96 h, animals were perfused and the brain was dissected for processing transverse and sagittal sections. Subsequently, fluorescent immunohistochemistry for GFP and Serotonin (5-hydroxytryptamine [5-HT]) was performed in the brain sections. Under the microscope, each RN subset was characterized individually from caudal to rostral according to the atlas. GFP+ or GFP/5-HT double labeled neurons in each subset were quantified for statistical analysis. RESULTS: At 72-h post-infection, very few GFP+ or GFP/5-HT double-labeled neurons appeared in the brainstem and beyond. In contrast, many labeled neurons were found at these levels after 96 h. Quantitative analysis showed that the majority of infected 5-HT+ neurons were located in the pallidus, obscurus, and magnus nuclei. Conversely, very few infected neurons were found in other raphe subsets, that is the pontis, median, dorsal, or linear nuclei. Overall, the raphe magnus had the highest number of GFP-labeled and GFP/5-HT double-labeled cells. CONCLUSIONS: The caudal subsets of RN, especially the raphe magnus, are the main sources of serotonergic input to the lower spinal cord controlling bladder activity.

Soluble TNFα Signaling within the Spinal Cord Contributes to the Development of Autonomic Dysreflexia and Ensuing Vascular and Immune Dysfunction after Spinal Cord Injury.

Cardiovascular disease and susceptibility to infection are leading causes of morbidity and mortality for individuals with spinal cord injury (SCI). A major contributor to these is autonomic dysreflexia (AD), an amplified reaction of the autonomic nervous system (hallmarked by severe hypertension) in response to sensory stimuli below the injury. Maladaptive plasticity of the spinal sympathetic reflex circuit below the SCI results in AD intensification over time. Mechanisms underlying this maladaptive plasticity are poorly understood, restricting the identification of treatments. Thus, no preventative treatments are currently available. Neuroinflammation has been implicated in other pathologies associated with hyperexcitable neural circuits. Specifically, the soluble form of TNFα (sTNFα) is known to play a role in neuroplasticity. We hypothesize that persistent expression of sTNFα in spinal cord underlies AD exacerbation. To test this, we intrathecally administered XPro1595, a biologic that renders sTNFα nonfunctional, after complete, high-level SCI in female rats. This dramatically attenuated the intensification of colorectal distension-induced and naturally occurring AD events. This improvement is mediated via decreased sprouting of nociceptive primary afferents and activation of the spinal sympathetic reflex circuit. We also examined peripheral vascular function using ex vivo pressurized arterial preparations and immune function via flow cytometric analysis of splenocytes. Diminishing AD via pharmacological inhibition of sTNFα mitigated ensuing vascular hypersensitivity and immune dysfunction. This is the first demonstration that neuroinflammation-induced sTNFα is critical for altering the spinal sympathetic reflex circuit, elucidating a novel mechanism for AD. Importantly, we identify the first potential pharmacological, prophylactic treatment for this life-threatening syndrome.SIGNIFICANCE STATEMENT Autonomic dysreflexia (AD), a disorder that develops after spinal cord injury (SCI) and is hallmarked by sudden, extreme hypertension, contributes to cardiovascular disease and susceptibility to infection, respectively, two leading causes of mortality and morbidity in SCI patients. We demonstrate that neuroinflammation-induced expression of soluble TNFα plays a critical role in AD, elucidating a novel underlying mechanism. We found that intrathecal administration after SCI of a biologic that inhibits soluble TNFα signaling dramatically attenuates AD and significantly reduces AD-associated peripheral vascular and immune dysfunction. We identified mechanisms behind diminished plasticity of neuronal populations within the spinal sympathetic reflex circuit. This study is the first to pinpoint a potential pharmacological, prophylactic strategy to attenuate AD and ensuing cardiovascular and immune dysfunction.