Multipotent bone marrow cell-seeded polymeric composites drive long-term, definitive urinary bladder tissue regeneration.

To date, there are no efficacious translational solutions for end-stage urinary bladder dysfunction. Current surgical strategies, including urinary diversion and bladder augmentation enterocystoplasty (BAE), utilize autologous intestinal segments (e.g. ileum) to increase bladder capacity to protect renal function. Considered the standard of care, BAE is fraught with numerous short- and long-term clinical complications. Previous clinical trials employing tissue engineering approaches for bladder tissue regeneration have also been unable to translate bench-top findings into clinical practice. Major obstacles still persist that need to be overcome in order to advance tissue-engineered products into the clinical arena. These include scaffold/bladder incongruencies, the acquisition and utility of appropriate cells for anatomic and physiologic tissue recapitulation, and the choice of an appropriate animal model for testing. In this study, we demonstrate that the elastomeric, bladder biomechanocompatible poly(1,8-octamethylene-citrate-co-octanol) (PRS; synthetic) scaffold coseeded with autologous bone marrow-derived mesenchymal stem cells and CD34+ hematopoietic stem/progenitor cells support robust long-term, functional bladder tissue regeneration within the context of a clinically relevant baboon bladder augmentation model simulating bladder trauma. Partially cystectomized baboons were independently augmented with either autologous ileum or stem-cell-seeded small-intestinal submucosa (SIS; a commercially available biological scaffold) or PRS grafts. Stem-cell synergism promoted functional trilayer bladder tissue regeneration, including whole-graft neurovascularization, in both cell-seeded grafts. However, PRS-augmented animals demonstrated fewer clinical complications and more advantageous tissue characterization metrics compared to ileum and SIS-augmented animals. Two-year study data demonstrate that PRS/stem-cell-seeded grafts drive bladder tissue regeneration and are a suitable alternative to BAE.

Mouse and Rat Anesthesia and Analgesia.

Anesthesia and analgesia play pivotal roles in ethically and humanely using animal models in research, especially concerning mice and rats. These rodent species, extensively utilized in scientific investigations due to their genetic resemblance to humans, serve as invaluable tools for studying diseases and testing treatments. Proper anesthesia and analgesia not only prioritize animal welfare but also heighten experimental validity by minimizing stress-induced physiological responses. Recent years have seen remarkable advancements in anesthesia for mice and rats. The focus has shifted away from the 'one size fits all' toward tailoring anesthesia protocols, considering factors like age, strain, and the nature of the experimental procedure. The use of inhalation agents such as isoflurane and sevoflurane is often preferred due to their rapid induction and recovery characteristics, allowing precise control over anesthesia depth. However, refinements in injectable anesthetic agents also provide researchers the flexibility to select suitable agents based on study requirements. Additionally, progress in analgesic techniques has led to effective pain management strategies for these rodents. Common analgesics such as nonsteroidal anti-inflammatory drugs (NSAIDs), opioids, and local anesthetics are administered to alleviate pain and discomfort. However, standard practice also involves continuous monitoring of animals' behavior and physiological parameters, ensuring timely adjustments in analgesic regimens for optimal pain relief without compromising experimental outcomes. By integrating tailored anesthesia and analgesia protocols into the experimental design, researchers uphold high animal welfare standards while obtaining reliable scientific data. This contributes significantly to advancing medical knowledge and therapeutic interventions with reproducible results. Published 2024. This article is a U.S. Government work and is in the public domain in the USA. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Injectable anesthesia for mouse and rat Basic Protocol 2: Inhalant anesthesia using isoflurane for mouse and rat Basic Protocol 3: Analgesia for mice and rats.

Etoposide-induced MicroRNA-205-5p Suppresses Proliferation and Migration by Targeting ERBB4 in MCF-7 Cells.

BACKGROUND/AIM: Patients with breast cancer frequently encounter a dismal prognosis due to the lack of effective and curative therapies. MicroRNAs (miRNAs) are aberrantly regulated in many types of cancer and have been recognized to play crucial roles in cancer progression. We performed a preclinical investigation of the anti-cancer effect of etoposide and microRNA-205-5p (miRNA-205-5p) and their relationship in MCF-7 cells. MATERIALS AND METHODS: Two cell culture systems, namely monolayers and spheroids, were employed for evaluating the effect of etoposide and miRNA-205-5p on cell proliferation and migration. Real time quantitative polymerase chain reaction was used for the measurement of mRNA and miRNA levels. Luciferase and western blot assays were utilized for the validation of the target gene of miRNA-205-5p. RESULTS: Treatment with etoposide, suppressed both cell proliferation and migration in MCF-7 monolayers. Also, the growth of MCF-7 spheroids as demonstrated by size measurements was inhibited by etoposide treatment. Furthermore, etoposide was found to upregulate the level of miRNA-205-5p. Over-expression of miRNA-205-5p inhibits cell proliferation and migration by directly targeting Erb-B2 receptor tyrosine kinase 4 (ERBB4). CONCLUSION: miRNA-205-5p may act as an important mediator of the anti-cancer effect of etoposide and miRNA-205-5p-based therapy may expand the therapeutic opportunities for breast cancer.

Histological Bulbar Manifestations in the ALS Rat.

BACKGROUND: Almost all patients with amyotrophic lateral sclerosis (ALS) develop bulbar symptoms; therefore, it is important to have valid animal models that accurately reflect these features. While the SOD1-G93A rat is extensively used as an ALS model, bulbar symptoms in this model are not well characterized. OBJECTIVE: In the present study, we aimed to better characterize bulbar dysfunction in terms of histology to determine whether the SOD1-G93A rat is a useful model for bulbar-onset ALS. METHODS: Sixty-day-old SOD1-G93A rats on a Sprague-Dawley background and age-matched wild-type controls were assessed weekly for global motor function, facial nerve function, and vagal nerve function. The study endpoint was determined when an SOD1-G93A rat could not right itself within 30 s of being placed on its side. At that point, neuronal counts were assessed in different brainstem cranial nerve nuclei. In addition, the masseter muscle, posterior belly of the digastric muscle, and tongue muscle were evaluated for intact neuromuscular junctions. RESULTS: Our data demonstrate decreases in the number of motor neurons in the trigeminal, facial, and hypoglossal nuclei, as well as compromised neuromuscular junction integrity in the muscles they innervate. CONCLUSION: These findings suggest that, from a histological standpoint, the SOD1-G93A rat is a valid model of ALS bulbar symptoms.

Spontaneous laryngeal reinnervation following chronic recurrent laryngeal nerve injury.

OBJECTIVES/HYPOTHESIS: To enhance understanding of spontaneous laryngeal muscle reinnervation following severe recurrent laryngeal nerve injury by testing the hypotheses that 1) nerve fibers responsible for thyroarytenoid muscle reinnervation can originate from multiple sources and 2) superior laryngeal nerve is a source of reinnervation. STUDY DESIGN: Prospective, controlled, animal model. METHODS: A combination of retrograde neuronal labeling techniques, immunohistochemistry, electromyography, and sequential observations of vocal fold mobility were employed in rat model of chronic recurrent laryngeal nerve injury. The current study details an initial set of experiments in sham surgical and denervated group animals and a subsequent set of experiments in a denervated group. RESULTS: At 3 months after recurrent laryngeal nerve resection, retrograde brainstem neuronal labeling identified cells in the characteristic superior laryngeal nerve cell body location as well as cells in a novel caudal location. Regrowth of neuron fibers across the site of previous recurrent laryngeal nerve resection was seen in 87% of examined animals in the denervated group. Electromyographic data support innervation by both the superior and recurrent laryngeal nerves following chronic recurrent laryngeal nerve injury. CONCLUSIONS: Following chronic recurrent laryngeal nerve injury in the rat, laryngeal innervation is demonstrated through the superior laryngeal nerve from cells both within and outside of the normal cluster of cells that supply the superior laryngeal nerve. The recurrent laryngeal nerve regenerates across a surgically created gap, but functional significance of regenerated nerve fibers is unclear.

Neuromuscular effects of G93A-SOD1 expression in zebrafish.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a fatal disorder involving the degeneration and loss of motor neurons. The mechanisms of motor neuron loss in ALS are unknown and there are no effective treatments. Defects in the distal axon and at the neuromuscular junction are early events in the disease course, and zebrafish provide a promising in vivo system to examine cellular mechanisms and treatments for these events in ALS pathogenesis. RESULTS: We demonstrate that transient genetic manipulation of zebrafish to express G93A-SOD1, a mutation associated with familial ALS, results in early defects in motor neuron outgrowth and axonal branching. This is consistent with previous reports on motor neuron axonal defects associated with familial ALS genes following knockdown or mutant protein overexpression. We also demonstrate that upregulation of growth factor signaling is capable of rescuing these early defects, validating the potential of the model for therapeutic discovery. We generated stable transgenic zebrafish lines expressing G93A-SOD1 to further characterize the consequences of G93A-SOD1 expression on neuromuscular pathology and disease progression. Behavioral monitoring reveals evidence of motor dysfunction and decreased activity in transgenic ALS zebrafish. Examination of neuromuscular and neuronal pathology throughout the disease course reveals a loss of neuromuscular junctions and alterations in motor neuron innervations patterns with disease progression. Finally, motor neuron cell loss is evident later in the disease. CONCLUSIONS: This sequence of events reflects the stepwise mechanisms of degeneration in ALS, and provides a novel model for mechanistic discovery and therapeutic development for neuromuscular degeneration in ALS.

Neuron-astrocyte signaling network in spinal cord dorsal horn mediates painful neuropathy of type 2 diabetes.

Activation of the neuronal-glial network in the spinal cord dorsal horn (SCDH) mediates various chronic painful conditions. We studied spinal neuronal-astrocyte signaling interactions involved in the maintenance of painful diabetic neuropathy (PDN) in type 2 diabetes. We used the db/db mouse, an animal model for PDN of type 2 diabetes, which develops mechanical allodynia from 6 to 12 wk of age. In this study, enhanced substance P expression was detected in the presynaptic sensory fibers innervating lamina I-III in the lumbar SCDH (LSCDH) of the db/db mouse at 10 wk of age. This phenomenon is associated with enhanced spinal ERK1/2 phosphorylation in projection sensory neurons and regional astrocyte activation. In addition, peak phosphorylation of the NR1 subunit of N-methyl-D-aspartate receptor (NMDAR), along with upregulation of neuronal and inducible nitric oxide synthase (nNOS and iNOS) expression were detected in diabetic mice. Expression of nNOS and iNOS was detected in both interneurons and astrocytes in lamina I-III of the LSCDH. Treatment with MK801, an NMDAR inhibitor, inhibited mechanical allodynia, ERK1/2 phosphorylation, and nNOS and iNOS upregulation in diabetic mice. MK801 also reduced astrocytosis and glial acidic fibrillary protein upregulation in db/db mice. In addition, N(G)-nitro-L-arginine methyl ester (L-NAME), a nonspecific NOS inhibitor, had similar effects on NMDAR signaling and NOS expression. These results suggest that nitric oxide from surrounding interneurons and astrocytes interacts with NMDAR-dependent signaling in the projection neurons of the SCDH during the maintenance of PDN.

Novel model to assess laryngeal function, innervation, and reinnervation.

OBJECTIVES: Laryngeal paralysis remains an unsolved problem, and the behavior of the laryngeal muscles following injury to the native innervation appears to be a function of denervation and reinnervation. The aim of the present study was to develop a reliable, accurate, and multifaceted animal model for study of laryngeal function, innervation, and reinnervation. METHODS: A spontaneous-breathing anesthesia technique, suspension laryngoscopy, endoscopic evaluation of the rat larynx, and transoral injection of a retrograde neuronal tracer, hydroxystilbamidine (FluoroGold), were developed. We submitted 14 rats to the developed technique to map the brain stem projections of the superior laryngeal nerve and the recurrent laryngeal nerve and to determine the feasibility and accuracy of the endoscopic injection technique. RESULTS: This endoscopic technique provided full evaluation of the rat larynx. We performed transoral endoscopic injection of FluoroGold and transcervical application of the tracer to transected superior laryngeal and recurrent laryngeal nerves in 14 different rats and successfully created a neural projection map for the superior laryngeal nerve, the recurrent laryngeal nerve, and the cervical ganglia. CONCLUSIONS: A reliable, accurate model for the characterization of laryngeal function, routes of innervation, and sources of spontaneous reinnervation following recurrent laryngeal nerve resection has been developed. This stable and reproducible model can serve as a dependable tool in future investigations of laryngeal nerve injury and recovery.

Transcriptional profiling of diabetic neuropathy in the BKS db/db mouse: a model of type 2 diabetes.

OBJECTIVE: A better understanding of the molecular mechanisms underlying the development and progression of diabetic neuropathy (DN) is essential for the design of mechanism-based therapies. We examined changes in global gene expression to define pathways regulated by diabetes in peripheral nerve. RESEARCH DESIGN AND METHODS: Microarray data for 24-week-old BKS db/db and db/+ mouse sciatic nerve were analyzed to define significantly differentially expressed genes (DEGs); DEGs were further analyzed to identify regulated biological processes and pathways. Expression profile clustering was performed to identify coexpressed DEGs. A set of coexpressed lipid metabolism genes was used for promoter sequence analysis. RESULTS: Gene expression changes are consistent with structural changes of axonal degeneration. Pathways regulated in the db/db nerve include lipid metabolism, carbohydrate metabolism, energy metabolism, peroxisome proliferator-activated receptor signaling, apoptosis, and axon guidance. Promoter sequences of lipid metabolism-related genes exhibit evidence of coregulation of lipid metabolism and nervous system development genes. CONCLUSIONS: Our data support existing hypotheses regarding hyperglycemia-mediated nerve damage in DN. Moreover, our analyses revealed a possible coregulation mechanism connecting hyperlipidemia and axonal degeneration.

The effects of anesthesia on measures of nerve conduction velocity in male C57Bl6/J mice.

Animal models, particularly mice, are used extensively to investigate neurological diseases. Basic research regarding animal models of human neurological disease requires that the animals exhibit hall mark characteristics of the disease. These include disease specific anatomical, metabolic and behavioral changes. Nerve conduction velocity (NCV) is the predominant method used to assess peripheral nerve health. Normative data adjusted for age, gender and height is available for human patients; however, these data are not available for most rodents including mice. NCV may be affected by animal age and size, body temperature, stimulus strength and anesthesia. While the effects of temperature, age and size are documented, the direct and indirect effects of anesthesia on NCV are not well reported. Our laboratory is primarily concerned with animal models of diabetic neuropathy (DN) and uses NCV to confirm the presence of neuropathy. To ensure that subtle changes in NCV are reliably assayed and not directly or indirectly affected by anesthesia, we compared the effects of 4 commonly used anesthetics, isoflurane, ketamine/xylazine, sodium pentobarbital and 2-2-2 tribromoethanol on NCV in a commonly used rodent model, the C57Bl6/J mouse. Our results indicate that of the anesthetics tested, isoflurane has minimal impact on NCV and is the safest, most effective method of anesthesia. Our data strongly suggest that isoflurane should become the anesthetic of choice when performing NCV on murine models of neurological disease.

p38 mediates mechanical allodynia in a mouse model of type 2 diabetes.

BACKGROUND: Painful Diabetic Neuropathy (PDN) affects more than 25% of patients with type 2 diabetes; however, the pathogenesis remains unclear due to lack of knowledge of the molecular mechanisms leading to PDN. In our current study, we use an animal model of type 2 diabetes in order to understand the roles of p38 in PDN. Previously, we have demonstrated that the C57BLK db/db (db/db) mouse, a model of type 2 diabetes that carries the loss-of-function leptin receptor mutant, develops mechanical allodynia in the hind paws during the early stage (6-12 wk of age) of diabetes. Using this timeline of PDN, we can investigate the signaling mechanisms underlying mechanical allodynia in the db/db mouse. RESULTS: We studied the role of p38 in lumbar dorsal root ganglia (LDRG) during the development of mechanical allodynia in db/db mice. p38 phosphorylation was detected by immunoblots at the early stage of mechanical allodynia in LDRG of diabetic mice. Phosphorylated p38 (pp38) immunoreactivity was detected mostly in the small- to medium-sized LDRG neurons during the time period of mechanical allodynia. Treatment with an antibody against nerve growth factor (NGF) significantly inhibited p38 phosphorylation in LDRG of diabetic mice. In addition, we detected higher levels of inflammatory mediators, including cyclooxygenase (COX) 2, inducible nitric oxide synthases (iNOS), and tumor necrosis factor (TNF)-alpha in LDRG neurons of db/db mice compared to non-diabetic db+ mice. Intrathecal delivery of SB203580, a p38 inhibitor, significantly inhibited the development of mechanical allodynia and the upregulation of COX2, iNOS and TNF-alpha. CONCLUSIONS: Our findings suggest that NGF activated-p38 phosphorylation mediates mechanical allodynia in the db/db mouse by upregulation of multiple inflammatory mediators in LDRG.

Lack of both bradykinin B1 and B2 receptors enhances nephropathy, neuropathy, and bone mineral loss in Akita diabetic mice.

An insertion polymorphism of the angiotensin-I converting enzyme gene (ACE) is common in humans and the higher expressing allele is associated with an increased risk of diabetic complications. The ACE polymorphism does not significantly affect blood pressure or angiotensin II levels, suggesting that the kallikrein-kinin system partly mediates the effects of the polymorphism. We have therefore explored the influence of lack of both bradykinin receptors (B1R and B2R) on diabetic nephropathy, neuropathy, and osteopathy in male mice heterozygous for the Akita diabetogenic mutation in the insulin 2 gene (Ins2). We find that all of the detrimental phenotypes observed in Akita diabetes are enhanced by lack of both B1R and B2R, including urinary albumin excretion, glomerulosclerosis, glomerular basement membrane thickening, mitochondrial DNA deletions, reduction of nerve conduction velocities and of heat sensation, and bone mineral loss. Absence of the bradykinin receptors also enhances the diabetes-associated increases in plasma thiobarbituric acid-reactive substances, mitochondrial DNA deletions, and renal expression of fibrogenic genes, including transforming growth factor beta1, connective tissue growth factor, and endothelin-1. Thus, lack of B1R and B2R exacerbates diabetic complications. The enhanced renal injury in diabetic mice caused by lack of B1R and B2R may be mediated by a combination of increases in oxidative stress, mitochondrial DNA damage and over expression of fibrogenic genes.

Intraspinal cord delivery of IGF-I mediated by adeno-associated virus 2 is neuroprotective in a rat model of familial ALS.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a devastating disease that is characterized by the progressive loss of motor neurons. Patients with ALS usually die from respiratory failure due to respiratory muscle paralysis. Consequently, therapies aimed at preserving segmental function of the respiratory motor neurons could extend life for these patients. Insulin-like growth factor-I (IGF-I) is known to be a potent survival factor for motor neurons. In this study we induced high levels of IGF-I expression in the cervical spinal cord of hSOD1(G93A) rats with intraspinal cord (ISC) injections of an adeno-associated virus serotype 2 vector (CERE-130). This approach reduced the extent of motor neuron loss in the treated segments of the spinal cord. However, a corresponding preservation of motor function was observed in male, but not female, hSOD1(G93A) rats. We conclude that ISC injection of CERE-130 has the potential to protect motor neurons and preserve neuromuscular function in ALS.