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Transplantation and repair: Combined cell implantation and chondroitinase delivery prevents deterioration of bladder function in rats with complete spinal cord injury
Abstract and Figures
Additional examination. In this study, we report changes in bladder function after a combined treatment that was designed to study axonal regeneration after complete spinal cord injury (SCI) in rats.
To report effects on bladder function following the administration of a combined treatment for complete SCI.
University of Alberta, Faculty of Rehabilitation Medicine, Edmonton, Canada.
Eight rats received Schwann cells in Matrigel-filled guidance channels, olfactory ensheathing glia and chondroitinase ABC at the lesion site following complete thoracic SCI. Controls (n=7) received Matrigel only. Daily bladder examinations were performed. Analysis of bladder size, wall thickness, actin and collagen type III was performed after 14 weeks.
Following SCI, both groups regained bladder voiding after 3 weeks. However, 2 weeks later, incontinence was observed in all untreated rats and two treated rats. Post-mortem examination of bladders revealed enlarged bladder sizes. Thicker bladder walls were found in untreated rats, which were composed of disorganized bundles of smooth muscle fibers surrounded by high amounts of collagen (type III).
We show that the combined treatment prevents collagen deposition in bladder walls and maintains the rat's ability to void efficiently. Although the mechanism responsible for this improvement is unclear, our study shows that the present combinatory therapy can influence bladder function, thus expanding their utility as a broad reparative approach for SCI.
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... Connective tissue fibril alignment can play a significant role in the mechanical properties of the bladder tissue [15], and increased collagen deposition within the bladder walls has been associated with non-compliant bladder function SCI in rodent models [16,17]. ...
Following spinal cord injury (SCI), pathological reflexes develop that result in altered bladder function and sphincter dis-coordination, with accompanying changes in the detrusor. Bladder chemodenervation is known to ablate the pathological reflexes, but the resultant effects on the bladder tissue are poorly defined. In a rodent model of contusion SCI, we examined the effect of early bladder chemodenervation with botulinum toxin A (BoNT-A) on bladder histopathology and collagen deposition. Adult female Long Evans rats were given a severe contusion SCI at spinal level T9. The SCI rats immediately underwent open laparotomy and received detrusor injections of either BoNT-A (10 U/animal) or saline. At eight weeks post injury, the bladders were collected, weighed, and examined histologically. BoNT-A injected bladders of SCI rats (SCI + BoNT-A) weighed significantly less than saline injected bladders of SCI rats (SCI + saline) (241 ± 25 mg vs. 183 ± 42 mg; p < 0.05). Histological analyses showed that SCI resulted in significantly thicker bladder walls due to detrusor hypertrophy and fibrosis compared to bladders from uninjured animals (339 ± 89.0 μm vs. 193 ± 47.9 μm; p < 0.0001). SCI + BoNT-A animals had significantly thinner bladder walls compared to SCI + saline animals (202 ± 55.4 μm vs. 339 ± 89.0 μm; p < 0.0001). SCI + BoNT-A animals had collagen organization in the bladder walls similar to that of uninjured animals. Detrusor chemodenervation soon after SCI appears to preserve bladder tissue integrity by reducing the development of detrusor fibrosis and hypertrophy associated with SCI.
... Connective tissue fibril alignment can play a significant role in the mechanical properties of the bladder tissue [15], and increased collagen deposition within the bladder walls has been associated with non-compliant bladder function SCI in rodent models [16,17]. Here, we showed that acute chemodenervation of the bladder with BoNT-A resulted in reduced collagen deposition and preserved collagen alignment within the lamina propria of the bladder, consistent with a more compliant bladder. ...
Following spinal cord injury (SCI), pathological reflexes develop that result in altered bladder function and sphincter dis-coordination, with accompanying changes in the detrusor. Bladder chemodenervation is known to ablate the pathological reflexes, but the resultant effects on the bladder tissue are poorly defined. In a rodent model of contusion SCI, we examined the effect of early bladder chemodenervation with botulinum toxin A (BoNT-A) on bladder histopathology and collagen deposition. Adult female Long Evans rats were given a severe contusion SCI at spi-nal level T9. The SCI rats immediately underwent open laparotomy and received detrusor injec-tions of either BoNT-A (10 U/animal) or saline. At 8 weeks post injury, the bladders were col-lected, weighed, and examined histologically. BoNT-A injected bladders of SCI rats (SCI-BoNT-A) weighed significantly less than saline injected bladders of SCI rats (SCI-saline) (241 ± 25 mg vs. 183 ± 42 mg; p<0.05). Histological analyses showed that SCI resulted in significantly thicker bladder walls due to detrusor hypertrophy and fibrosis compared to bladders from uninjured animals (339 ± 89.0 m vs. 193 ± 47.9 m; p<0.0001). SCI-BoNT-A animals had significantly thinner bladder walls compared to SCI-saline animals (202 ± 55.4 m vs. 339 ± 89.0 m; p<0.0001). SCI-BoNT-A animals had collagen organization in the bladder walls similar to that of uninjured animals. Detrusor chemodenervation soon after SCI appears to preserve bladder tissue integrity, by reducing the development of detrusor fibrosis and hypertrophy associated with SCI.
... Therefore, combinatorial treatment of SC transplantation and OPN in SCI models would likely be more effective promoting corticospinal tractdependent functional restoration in adults. In addition to OPN, SC transplantation in combination with other treatments also improved SC survival and migration within the injured spinal cord, and increased the axonal growth capacity, which enhanced axon (including corticospinal axon) regeneration into SC bridges beyond the caudal interface; this was accompanied by functional improvement (Pearse et al., 2004b;Fouad et al., 2009;Bunge and Wood, 2012;Ghosh et al., 2012;Wiliams and Bunge, 2012;Bunge, 2016). Given the disadvantages of SC transplantation alone, as well as the multifaceted pathophysiologic changes of SCI, combinatorial therapeutic strategies for SCI repair are necessary. ...
Spinal cord injury (SCI) can result in sensorimotor impairments or disability. Studies of the cellular response to SCI have increased our understanding of nerve regenerative failure following spinal cord trauma. Biological, engineering and rehabilitation strategies for repairing the injured spinal cord have shown impressive results in SCI models of both rodents and non-human primates. Cell transplantation, in particular, is becoming a highly promising approach due to the cells’ capacity to provide multiple benefits at the molecular, cellular, and circuit levels. While various cell types have been investigated, we focus on the use of Schwann cells (SCs) to promote SCI repair in this review. Transplantation of SCs promotes functional recovery in animal models and is safe for use in humans with subacute SCI. The rationales for the therapeutic use of SCs for SCI include enhancement of axon regeneration, remyelination of newborn or sparing axons, regulation of the inflammatory response, and maintenance of the survival of damaged tissue. However, little is known about the molecular mechanisms by which transplanted SCs exert a reparative effect on SCI. Moreover, SC-based therapeutic strategies face considerable challenges in preclinical studies. These issues must be clarified to make SC transplantation a feasible clinical option. In this review, we summarize the recent advances in SC transplantation for SCI, and highlight proposed mechanisms and challenges of SC-mediated therapy. The sparse information available on SC clinical application in patients with SCI is also discussed.
... Another approach which has been widely explored, is to combine OEC transplantation with additional exogenous molecules or with biomaterials. The first reported studies investigated the behaviors of OECs with chondroitinase of cAMP in vivo after SCI [182,183]. These studies report additional functional benefits in the co-grafted groups. ...
The primary olfactory system (POS) is in permanent renewal, especially the primary olfactory neurons (PON) are renewed with a turnover of around four weeks, even in adulthood. The re-growth of these axons is helped by a specific population of glial cells: the olfactory ensheathing cells (OECs). In the POS, OECs constitute an “open-channel” in which the axons of PON cause regrowth from peripheral nervous system (PNS) to central nervous system (CNS). The remarkable role played by OECs into the POS has led scientists to investigate their properties and potential beneficial effects after transplantation in different lesion models of the CNS and PNS. In this review, we will resume and discuss more than thirty years of research regarding OEC studies. Indeed, after discussing the embryonic origins of OECs, we will describe the in vitro and in vivo properties exert at physiological state by these cells. Thereafter, we will present and talk over the effects of the transplantation of OECs after spinal cord injury, peripheral injury and other CNS injury models such as demyelinating diseases or traumatic brain injury. Finally, the mechanisms exerted by OECs in these different CNS and PNS lesion paradigms will be stated and we will conclude by presenting the innovations and future directions which can be considered to improve OECs properties and allow us to envisage their use in the near future in clinical applications.
... 58 In rodents, SCI initially results in a period of bladder areflexia characterized by the presence of large residual urine volumes that cause bladder overdistension, which is usually resolved after approximately the first two weeks after SCI, when the lesioned animals recover voiding control. 59 In this regard, we found that the overexpression of Wnt5a also significantly delayed bladder function recovery after SCI. Although we cannot conclude the specific cellular and/or molecular mechanisms underlying the impairment in functional recovery observed due to Wnt5a overexpression, it should be noted that different reports have shown a consistent relationship between the preservation and function of the descending serotonergic system and the recovery of interlimb coordination and bladder function after SCI. ...
Accordingly to its known function in corticospinal tract (CST) developmental growth, previous reports have shown an inhibitory role of Wnt5a in CST regeneration after spinal cord injury (SCI). Interestingly, it has been subsequently demonstrated that Wnt5a also modulates the developmental growth of non‐CST axons and that different Wnt5a receptors are expressed in neurons, oligodendrocytes, NG2+ glial precursors and reactive microglia/macrophages and astrocytes after SCI. However, the role of Wnt5a in the response of these cell types, in the regeneration of non‐CST axons and in functional recovery after SCI is currently unknown. To evaluate this, rats were subjected to spinal cord contusion and injected with a lentiviral vector generated to overexpress Wnt5a. Histological analyses were performed in spinal cord sections processed for the visualization of myelin, oligodendrocytes, neurons, microglia/macrophages, astrocytes, NG2+ glial precursors and serotonergic axons. Motor and bladder function recovery were also assessed. Further advancing our knowledge on the role of Wnt5a in SCI, we found that, besides its previously reported functions, Wnt5a overexpression elicits a reduction on neuronal cell density, the accumulation of NG2+ glial precursors and the descending serotonergic innervation in the affected areas, along with impairment of motor and bladder function recovery after SCI.
Three theories of regeneration dominate neuroscience today, all purporting to explain why the adult central nervous system (CNS) cannot regenerate. One theory proposes that Nogo, a molecule expressed by myelin, prevents axonal growth. The second theory emphasizes the role of glial scars. The third theory proposes that chondroitin sulfate proteoglycans (CSPGs) prevent axon growth. Blockade of Nogo, CSPG, and their receptors indeed can stop axon growth in vitro and improve functional recovery in animal spinal cord injury (SCI) models. These therapies also increase sprouting of surviving axons and plasticity. However, many investigators have reported regenerating spinal tracts without eliminating Nogo, glial scar, or CSPG. For example, many motor and sensory axons grow spontaneously in contused spinal cords, crossing gliotic tissue and white matter surrounding the injury site. Sensory axons grow long distances in injured dorsal columns after peripheral nerve lesions. Cell transplants and treatments that increase cAMP and neurotrophins stimulate motor and sensory axons to cross glial scars and to grow long distances in white matter. Genetic studies deleting all members of the Nogo family and even the Nogo receptor do not always improve regeneration in mice. A recent study reported that suppressing the phosphatase and tensin homolog (PTEN) gene promotes prolific corticospinal tract regeneration. These findings cannot be explained by the current theories proposing that Nogo and glial scars prevent regeneration. Spinal axons clearly can and will grow through glial scars and Nogo-expressing tissue under some circumstances. The observation that deleting PTEN allows corticospinal tract regeneration indicates that the PTEN/AKT/mTOR pathway regulates axonal growth. Finally, many other factors stimulate spinal axonal growth, including conditioning lesions, cAMP, glycogen synthetase kinase inhibition, and neurotrophins. To explain these disparate regenerative phenomena, I propose that the spinal cord has evolved regenerative mechanisms that are normally suppressed by multiple extrinsic and intrinsic factors but can be activated by injury, mediated by the PTEN/AKT/mTOR, cAMP, and GSK3b pathways, to stimulate neural growth and proliferation.
Synthetic hydrogels composed of polymer pore frames are commonly used in medicine, from pharmacologically targeted drug delivery to the creation of bioengineering constructions used in implantation surgery. Among various possible materials, the most common are poly-[N(2-hydroxypropyl)methacrylamide] (pHPMA) derivatives. One of the pHPMA derivatives is biocompatible hydrogel, NeuroGel. Upon contact with nervous tissue, the NeuroGel's structure can support the chemical and physiological conditions of the tissue necessary for the growth of native cells. Owing to the different pore diameters in the hydrogel, not only macromolecules, but also cells can migrate. This study evaluated the differentiation of bone marrow stromal cells (BMSCs) into neurons, as well as the effectiveness of using this biofabricated system in spinal cord injury in vivo. The hydrogel was populated with BMSCs by injection or rehydration. After cultivation, these fragments (hydrogel + BMSCs) were implanted into the injured rat spinal cord. Fragments were immunostained before implantation and seven months after implantation. During cultivation with the hydrogel, both variants (injection/rehydration) of the BMSCs culture retained their viability and demonstrated a significant number of Ki-67-positive cells, indicating the preservation of their proliferative activity. In hydrogel fragments, BMSCs also maintained their viability during the period of cocultivation and were Ki-67-positive, but in significantly fewer numbers than in the cell culture. In addition, in fragments of hydrogel with grafted BMSCs, both by the injection or rehydration versions, we observed a significant number up to 57%–63.5% of NeuN-positive cells. These results suggest that the heterogeneous pHPMA hydrogel promotes neuronal differentiation of bone marrow-derived stromal cells. Furthermore, these data demonstrate the possible use of NeuroGel implants with grafted BMSCs for implantation into damaged areas of the spinal cord, with subsequent nerve fiber germination, nerve cell regeneration, and damaged segment restoration.
Spinal cord injury (SCI) can cause chronic paralysis and incontinence and remains a major worldwide healthcare burden, with no regenerative treatment clinically available. Intraspinal transplantation of olfactory ensheathing cells (OECs) and injection of chondroitinase ABC (chABC) are both promising therapies but limited and unpredictable responses are seen, particularly in canine clinical trials. Sustained delivery of chABC presents a challenge due to its thermal instability; we hypothesised that transplantation of canine olfactory mucosal OECs genetically modified ex vivo by lentiviral transduction to express chABC (cOEC-chABC) would provide novel delivery of chABC and synergistic therapy. Rats were randomly divided into cOEC-chABC, cOEC, or vehicle transplanted groups and received transplant immediately after dorsal column crush corticospinal tract (CST) injury. Rehabilitation for forepaw reaching and blinded behavioural testing was conducted for 8 weeks. We show that cOEC-chABC transplanted animals recover greater forepaw reaching accuracy on Whishaw testing and more normal gait than cOEC transplanted or vehicle control rats. Increased CST axon sprouting cranial to the injury and serotonergic fibres caudal to the injury suggest a mechanism for recovery. We therefore demonstrate that cOECs can deliver sufficient chABC to drive modest functional improvement, and that this genetically engineered cellular and molecular approach is a feasible combination therapy for SCI.
Objective:
To provide an up-to-date review of studies that used preclinical animal models for the evaluation of tissue engineering treatments for spinal cord injury (SCI), which involved the use of biomaterials with or without the addition of cells or biomolecules.
Methods:
Electronic search of the PubMed, Web of Science and Embase databases was performed for relevant studies published between January 2009 and December 2019.
Results:
1579 articles were retrieved, of which 58 studies were included for analysis. Among the included studies, rats were the most common species used for animal models of SCI, while complete transection was the most commonly used injury pattern. Immediate intervention after injury was conducted in the majority of studies, and 8 weeks was the most common final time point of outcome assessment. A wide range of natural and synthetic biomaterials with different morphologies were used as a part of tissue engineering treatments for SCI, including scaffolds, hydrogels and particles.
Conclusion:
Experimental parameters in studies using SCI animal models to evaluate tissue engineering treatments should be carefully considered to match the purpose of the study. Biomaterials that have functional modifications or are applied in combination with cells and biomolecules can be effective in creating a permissive environment for SCI repair in preclinical animal models.
Transplantation of olfactory ensheathing cells, the glia of the primary olfactory nervous system, has been trialed for spinal cord injury repair with promising but variable outcomes in animals and humans. Olfactory ensheathing cells can be harvested either from the lamina propria beneath the neuroepithelium in the nasal cavity, or from the olfactory bulb in the brain. As these areas contain several other cell types, isolating and purifying olfactory ensheathing cells is a critical part of the process. It is largely unknown how contaminating cells such as fibroblasts, other glial cell types and supporting cells affect olfactory ensheathing cell function post-transplantation; these cells may also cause unwanted side-effects. It is also, however, possible that the presence of some of the contaminant cells can improve outcomes. Here, we reviewed the last decade of olfactory ensheathing cell transplantation studies in rodents, with a focus on olfactory ensheathing cell purity. We analyzed how purification methods and resultant cell purity differed between olfactory mucosa- and olfactory bulb-derived cell preparations. We analyzed how the studies reported on olfactory ensheathing cell purity and which criteria were used to define cells as olfactory ensheathing cells. Finally, we analyzed the correlation between cell purity and transplantation outcomes. We found that olfactory bulb-derived olfactory ensheathing cell preparations are typically purer than mucosa-derived preparations. We concluded that there is an association between high olfactory ensheathing cell purity and favourable outcomes, but the lack of olfactory ensheathing cell-specific markers severely hampers the field.
Intravenous administration of the selective 5-hydroxytryptamine (5-HT)1A receptor agonist 8-hydroxy-2-(di-N-propylaminotetralin (8-OH-DPAT) and of a low doses of buspirone elicited the supraspinal micturition reflex (SMR) in urethane-anesthetized rats when the urinary bladder was filled with just a subthreshold volume of saline (threshold conditions). The effect of i.v. 8-OH-DPAT was abolished by hexamethonium or spiroxatrine. When SMR was elicited by bladder distension (suprathreshold conditions), i.v. 8-OH-DPAT increased the frequency of bladder contractions. In threshold conditions, stimulation of SMR was also induced by i.c.v. or by i.t. administration of 8-OH-DPAT and 5-HT but not by topical application of 8-OH-DPAT onto the bladder. Guanethidine pretreatment, which produced detrusor hyperreflexia, antagonized the effect of both i.c.v. and i.t. 8-OH-DPAT. In rats treated with capsaicin as adults, the response to 8-OH-DPAT was unchanged. In rats treated with capsaicin as newborns, instead, the response to i.t. 8-OH-DPAT was abolished and that to i.c.v. 8-OH-DPAT was shifted to higher doses. Pretreatment with 5,7-dihyroxytryptamine did not affect the response to i.t. 8-OH-DPAT but shifted to higher doses the response to i.c.v. 8-OH-DPAT. Intravenous administration of spiroxatrine, methysergide, NAN-190 [1-(2-methoxyphenyl)-4-[4-(2-phtalimido)butyl] piperazine] or high doses of buspirone but not of 1-sulpiride inhibited SMR in suprathreshold conditions. The inhibitory effect of spiroxatrine, NAN-190 and buspirone was not reduced by guanethidine pretreatment. In chronically spinalized animals, i.v. 8-OH-DPAT increased the amplitude of the reflex bladder contractions induced by bladder distension.(ABSTRACT TRUNCATED AT 250 WORDS)
Electrophysiological techniques were used to examine the organization of the spinobulbospinal micturition reflex pathway in the rat. Electrical stimulation of afferent axons in the pelvic nerve evoked a long latency (136 +/- 41 ms) response on bladder postganglionic nerves, whereas stimulation in the dorsal pontine tegmentum elicited shorter latency firing (72 +/- 25 ms) on these nerves. Transection of the pelvic nerve eliminated these responses. Firing on the bladder postganglionic nerves was evoked by stimulation in a relatively limited area of the pons within and close to the laterodorsal tegmental nucleus (LDT) and adjacent ventral periaqueductal gray. Stimulation at sites ventral to this excitatory area inhibited at latencies of 107 +/- 11 ms the asynchronous firing on the bladder postganglionic nerves elicited by bladder distension. Electrical stimulation of afferents in the pelvic nerve evoked short latency (13 +/- 3 ms) negative field potentials in the dorsal part of the periaqueductal gray as well as long latency (42 +/- 7 ms) field potentials in and adjacent to the LDT. The responses were not altered by neuromuscular blockade. Similar responses were elicited by stimulation of afferent axons in the bladder nerves. The sum of the latencies of the ascending and descending pathways between the LDT and the pelvic nerve (i.e. 72 ms plus 42 ms = 114 ms) is comparable although somewhat shorter (22 ms) than the latency of the entire micturition reflex. These results provide further evidence that the micturition reflex in the rat is mediated by a spinobulbospinal pathway which passes through the dorsal pontine tegmentum, and that neurons in the periaqueductal gray as well as the LDT may play as important role in the regulation of the micturition.
Micturition in cats and rats with an intact neuraxis is dependent upon a spinobulbospinal reflex activated by A delta bladder afferents. This report describes changes in micturition reflexes 2 h to 14 weeks following spinal cord transection at the lower thoracic level. In acute spinal cats micturition reflexes were blocked, however, several weeks after transection, a long latency (180-200 ms) spinal reflex could be activated by C-fiber bladder afferents. This reflex was blocked by capsaicin in doses (20-30 mg/kg, s.c.) that did not affect micturition reflexes in intact cats. Micturition reflexes were unmasked in acute spinal and facilitated in chronic spinal cats by naloxone, an opioid antagonist. Spinal neurons and axons containing opioid peptides were more prominent below the level of transection in chronic spinal cats. VIP, a putative neurotransmitter in C-fiber bladder afferents, inhibited micturition reflexes when injected intrathecally (2-10 micrograms) in intact cats but facilitated micturition reflexes in spinal cats (doses 0.1-1 micrograms, i.t.). VIP-containing C-fiber afferent projections to lamina I of the sacral spinal cord expanded in spinal cats. Thus VIP afferents may have an important role in the recovery of bladder reflexes after spinal injury. Paraplegic animals also exhibit bladder-sphincter dyssynergia, which causes functional outlet obstruction. Studies in rats have revealed that outlet obstruction induced by partial urethral ligation facilitates spinal micturition reflex pathways and causes an expansion of HRP-labelled bladder afferent projections in the spinal cord. These findings raise the possibility that the alterations in central reflex connections in paraplegic animals may be induced in part by changes in peripheral afferent input secondary to outlet obstruction.(ABSTRACT TRUNCATED AT 250 WORDS)
An immunohistochemically derived morphological description of a diverse population of rat lamina VII and X intraspinal 5HT neurons is provided. These bipolar or multipolar neurons occur most frequently in lamina X, dorsal or dorsolateral to the central canal, in thoracolumbar, sacral, and coccygeal spinal segments. These 5HT intraspinal neurons are found in normal rat spinal cords as well as in spinal cords that have been hemisected or transected 60 days prior to serotonin immunostaining. Therefore, 5HT intraspinal neurons are the probable source of the biochemically detectable 5HT that remains in the spinal cord distal to a spinal transection. In the rat, serotonin intraspinal neurons are most often associated with spinal autonomic nuclei but it is unknown if they are preganglionic in nature.
(1)The brain stem of anaesthetized cats has been mapped between Horsley-Clark planes APO and P8.5 with electrical stimuli of low intensity in order to determine the areas which can produce excitatory of inhibitory influences on the spontaneously contracting, sympathetically denervated, urinary bladder. (2) Two inhibitory areas were found. The first extended from P3.0 to P8.5 and at all levels was coincident with the midline raphe nuclei. The second area occurred largely 2-3 mm lateral to the midline, in the area of the nucleus reticularis pontis caudalis rostrally, and the nuclei reticularis gigantocellularis and parvocellularis caudally. Both of these areas were found to inhibit bladder contractions with threshold stimulus parameters of 20-60 microamperemeter, 400 microseconds, 20 Hz. (3)One excitatory area was found, largely 3-4 mm lateral to the midline. This area appeared large and diffuse in the lateral reticular formation. It is possible that it originated in the pontine micturition centre in the rostral pontine tegmentum. Caudally, it shifted to occupy a ventrolateral position. This excitatory area was in close approximation to, and was probably interspersed with, the lateral inhibitory area. (4) In decerebrate preparations the areas that produced excitation or inhibition had the same distribution as those found in anaesthetized animals. (5) Single shock stimulation (100 microamperemeter, 400 microseconds, 0.5 Hz repetition frequency in the excitatory area could produce firing in pelvic nerve efferents to the bladder at latencies of 60-110 ms. The amplitude of such responses was dependent on the level of intravesical pressure. (6) Stimulation of the inhibitory areas produced no evoked responses in the pelvic nerve efferents, but could inhibit reflexly evoked responses in this nerve. The similarity in the time courses of the inhibitory effects from the two areas raises the possibility that one acts via the other.
Collagen content was determined in the detrusor muscle from control rats and rats subjected to infravesical outflow obstruction for periods of 3 days to 4 months. During the 1st 6 weeks of obstruction the detrusor weight increased 12-fold. The total amount of detrusor collagen increased by a factor of 4, while the concentration of collagen decreased to 1/3 of the initial concentration. A longer period of obstruction, 4 months, did not further affect detrusor weight or collagen content or concentration. Ultrastructurally, both normal and hypertrophic detrusor showed a great number of collagen fibrils (probably synthetized by fibrocytes) between the smooth muscle bundles. Collagen fibrils (probably synthesized by the smooth muscle cells) could also, although less frequently, be found within the bundles. The decreased collagen concentration in the hypertrophic detrusor could largely be explained by a coalescence of the smooth muscle bundles resulting in a relative increase of this tissue component.
Spinal cord injury (SCI) at Th13 was induced in female Wistar rats, and changes in the urinary bladder were examined during the acute phase of SCI. Wet weights of the spinal bladders increased twofold over controls by 7 days after SCI. Intravesical volumes increased sixfold over control values by day 3, and then decreased 7 days after the injury. Maximal pressure within the bladder decreased in all spinal rats compared with controls. Smooth muscle cells were isolated from the urinary bladder, and their total protein and DNA content were measured by multiparametric cytofluorometry. DNA content of isolated smooth muscle cells decreased by day 3 and remained 7 days after the spinal injury. Total protein content of isolated smooth muscle cells was decreased 1 day after and increased 7 days after the spinal injury, just when spinal reflex of the bladder recovered. These findings suggest that hypertrophy of smooth muscle cells in urinary bladder is related to the activity of peripheral autonomic nerve and that smooth muscle cells already begin to hypertrophy during the spinal shock period to adjust themselves to the new state, that is, the spinal bladder.
Transneuronal tracing techniques were used to identify the putative spinal and brainstem neurons involved in continence and voiding in the female rat. Pseudorabies virus, Bartha's K strain, was injected into either the external urethral sphincter, the bladder base, or the bladder body. After 3-5 days, the rats were perfused with fixative, and virus-labelled cells were identified by using immunohistochemistry. External urethral sphincter (EUS) injections resulted in labelling of pudendal motoneurons in the dorsolateral nucleus of L6. Putative spinal interneurons were found in the medial cord from T13 to S1 and in the lateral gray of T13-L2 and L5-S1. After both bladder base and bladder body injections, the majority of pseudorabies virus-labelled cells were found in the lateral gray and medial cord of L6-S1. A number of those found in the intermediolateral cell column resembled the parasympathetic preganglionic neurons; the remaining neurons in the lateral and medial gray were presumed to be interneurons. Very few pseudorabies virus-labelled cells were found rostral to T10. In the brainstem, transneuronally labelled cells were found in the parapyramidal medullary reticular formation, Barrington's nucleus, raphe magnus, raphe pallidus, subcoeruleus pars alpha, locus coeruleus, the A5 noradrenergic cell group, and ventromedial periaqueductal gray after all injection sites. Pseudorabies virus-labelled cells were also seen in the forebrain following the longest survival times; areas consistently labelled included the lateral hypothalamus, the parvocellular region of the paraventricular nucleus, and the medial preoptic area. These studies indicate that there is a substantial overlap of central nervous system neurons that innervate the EUS and the bladder in the female.
Purpose:
Fibrosis of bladder tissue is characterized by an abnormal deposition of connective tissue within different layers of the bladder wall, resulting in a low volume, high pressure vesical which may ultimately contribute to renal scarring and failure. These bladders are functionally referred to as "non-compliant" and may result from different etiologies: neurogenic, which encompasses myelodysplasia and spinal cord injury, or non-neurogenic, owing to obstruction or radiation therapy. To examine the molecular mechanisms responsible for this fibrosis, we have analyzed a well-characterized pediatric patient population for alteration(s) in collagen types I and III regulation at the protein and nucleic acid levels.
Materials and methods:
Immunohistochemical localization of collagen subtypes (I, III, and IV) was carried out using type specific monoclonal antibodies. Total collagen was determined by hydroxyproline analysis, and subtype specific expression of collagenous proteins, following cyanogen bromide extraction procedures, was quantified by competitive ELISA. Total RNA was extracted by guanidinium/phenol/chloroform, and slot blot hybridization analyses with radiolabeled human cDNA probes were quantified by densitometry of resulting autoradiograms.
Results:
Connective tissue infiltration of detrusor smooth muscle bundles was specific for type III collagen. Protein analyses demonstrated: 1) an increase in total collagen, 2) a statistically significant increase in the type III: type I collagen ratio, and 3), an absolute increase in type III collagen protein in non-compliant bladder tissue. At the mRNA level, there was a coordinate increase in both collagen I and III steady-state mRNAs in non-neurogenic bladder tissue, whereas neurogenic bladder tissue was characterized by an increase in the type III: type I mRNA transcript ratio.
Conclusions:
These data suggest that regulation of collagen synthesis in bladder fibrosis is complex and is characterized by both transcriptional and post-transcriptional mechanisms, depending upon the etiology of the fibrosis.
To determine the feasibility of urodynamic monitoring of voiding function in conscious, female spinal cord-injured (SCI) rats and to compare the voiding function in SCI rats and those with normal spinal cord (NSC).
Cystometrograms were performed on conscious, female Sprague-Dawley rats. Parameters measured included voided volume, residual volume, volume threshold for inducing micturition, voiding efficiency, micturition pressure, pressure threshold for inducing micturition, and bladder contraction duration. SCI animals were studied 2 to 3 weeks after T8-T10 spinal cord transection.
Approximately one half of SCI rats exhibited uninhibited bladder contractions before voiding. These contractions were not observed in NSC rats. Compared with NSC rats, SCI rats had larger volume thresholds (1.43 versus 0.34 mL, P <0.001) and voided volumes (0.72 versus 0.31 mL, P <0.01). Although SCI rats had larger micturition pressures (65 versus 35 cm H2O, P <0.001), residual volumes were increased (0.71 versus 0.03 mL, P <0.001) and voiding efficiency was decreased (50% versus 92%, P <0.001) compared with the measurements in NSC rats. The micturition pressure threshold was slightly lower (23%, P <0.05) and the bladder contraction duration was longer (33%, P <0.05) in SCI rats than in NSC rats.
Cystometric studies in conscious female SCI and NSC rats revealed significant changes in the activity of the lower urinary tract after spinal cord transection. Fifty-five percent of SCI rats exhibited detrusor hyperreflexia during bladder filling and decreased voiding efficiency compared with NSC animals. This method of cystometric evaluation in the conscious animal is likely to be useful for evaluating new pharmacologic treatments for neurogenic bladder dysfunction.