Nerve fibers in tumors of the human urinary bladder

Abstract.Exophytic tumors of the urinary bladder were examined by means of transmission electron microscopy for the presence of neural tissue because, as yet, there has been hardly any discussion of a neuronal component in the biology of neoplasms. In the stroma and rarely in the epithelium of bladder tumors, fine nerve strands were found to be irregularly distributed. These strands comprised one to a maximum of five axons containing predominantly colocalized clear and dense-core vesicles. Immunohistochemistry revealed some nerve-like structures showing vasoactive intestinal neuropeptide (VIP) reactivity. This response and the combination of vesicle types indicate that parasympathetic cholinergic neurons contribute to the innervation of the tumors. Thus, a morphological basis for neuronal influence on the behavior of such tumors has been demonstrated.     

Morphology and distribution of dopaminergic neurons intrinsic to the human striatum

The putative dopaminergic (DA) neurons intrinsic to the human striatum were studied by applying immunofluorescence and quantitative methods to postmortem tissue from seven normal individuals. Stringent morphological and chemical criteria were used to identify striatal DA neurons, including immunostaining for tyrosine hydroxylase, DA transporter and neuronal nuclear protein. The DA neurons were scattered throughout the striatum, but abounded particularly in its ventral portion. Frequency distribution of surface areas of DA cell bodies reveals that the most frequent DA neurons (x =58.0%, S.D.=12.8%) had a medium-sized (approximately 200+/-15 microm2) perikaryon with 3-5 varicose dendrites, whereas others (x =35.5%, S.D.=14.0%) had a smaller (approximately 140+/-15 microm2) perikaryon with 3-4 varicose dendrites. There was a small number (x =6.5%, S.D.=8.5%) of larger DA neurons (209-584 microm2) with spiny dendrites and a few TH-immunoreactive cells displaying mixed neuron-glia morphology. Despite significant inter-individual variations in neuron density, the human striatum (mean volume of 8.76 cm3) harbored a mean of 331.9 DA neurons (S.D.=199.2). A prolific zone, containing about 3000 cells, occurred in the ventral striatum in two brains. The addition of these cells would increase by about 10 times the total number of striatal DA neurons, which should not be confounded with segments of nigrostriatal DA fibers that displayed large (8-12 microm) varicosities and looked like small bipolar neurons. The function of striatal DA neurons is unknown but the fact that their number increases markedly following lesion of nigral DA input or administration of various growth factors, opens up new therapeutic avenues for treatment of Parkinson's disease.     

Genetic modeling of human urinary bladder carcinogenesis

We developed a model of human urinary bladder cancer progression from in situ precursor lesions to invasive carcinoma using whole organ histologic and genetic mapping. The model represents a high-density and detailed analysis regarding allelic losses on chromosomes 4, 8, 9, 11, and 17 as revealed by testing of 234 samples obtained from five cystectomy specimens. The samples corresponded to microscopically identified intraurothelial precursor conditions ranging from dysplasia to carcinoma in situ and invasive cancer. The initial analysis of paired normal and tumor DNA samples disclosed allelic losses in 72 of 225 tested hypervariable DNA markers. Subsequent use of these markers on all mucosal samples revealed that 47 had alterations with a statistically significant relation to urothelial neoplasia. The allelic losses clustered in 33 distinct chromosomal regions, indicating the location of putative tumor suppressor genes involved in the development and progression of urinary bladder cancer. Some of the markers with statistically significant allelic losses mapped to the regions containing well-characterized tumor suppressor genes but many were located in previously unknown loci. The majority of statistically significant allelic losses (70%) occurred early in low-grade intraurothelial dysplasia, and some of them involved adjacent areas of morphologically normal mucosa preceding the development of microscopically recognizable precursor lesions. The remaining 30% of markers developed allelic losses in the later phases of urothelial neoplasia, implicating their involvement in progression to invasive disease. Markers exhibiting allelic losses in early phases of urothelial neoplasia could be used for detection of occult preclinical or even premicroscopic phases of urinary bladder cancer, whereas markers that showed allelic losses in the later phases of the process could serve as indicators of progression to invasive disease. The approach used in this study facilitates genome-wide modeling of cancer progression and provides important chromosomal landmarks for more specific studies of multistep urinary bladder carcinogenesis.     

Pathology of intrinsic cardiac neurons from ischemic human hearts

Various populations of intrinsic cardiac neurons influence regional cardiac function tonically. It is not known whether such neurons are affected by disease states and, if so, in what manner. Therefore, the morphology of intrinsic cardiac ganglia obtained from patients with angiographic evidence of compromised regional coronary blood supply was studied. Posterior atrial ganglia and surrounding fat, removed at the time of cardiac surgery, were placed immediately in saline and within 15-120 min (average of about 40 min) in 0.5% paraformaldehyde/2.5% glutaraldehyde. In 32 studied ganglia, 35% of 473 intrinsic cardiac neurons displayed striking pathological changes at the light and ultrastructural level. The other cells displayed normal morphology. The cytoplasm of 74% of the abnormal cells had one or more of three types of inclusions: (1) darkly stained lamellated inclusions (Type I), (2) membrane-bound whorls and parallel arrays of lightly stained membranes, as well as fine granular material (Type II), or (3) concentric layers of lightly stained membranes with a darker, granular core (Type III). Neurons with inclusions were markedly enlarged (66 x 54 microm vs. 40 x 34 microm for normal neurons) and displayed fewer dendrites. Some neurons contained electron lucent vacuoles indicative of degeneration while others showed frank degeneration, being fragmented, shrunken, and misshapen. Phagocytic cells containing lamellated inclusions and cellular debris were found in ganglia with abnormal neurons. Some axon terminals also displayed degenerative changes. The identification of pathological changes in the human intrinsic cardiac nervous system has implications with respect to the functional integrity of this final common regulator of cardiac function in disease states.     

Immunodetection of androgen receptor in human urinary bladder cancer

We investigated the expression of androgen receptor (AR) protein in transitional cell carcinoma of human urinary bladder in paraffin-embedded sections of tumours obtained from nine patients with urinary bladder cancer treated by radical cystectomy. In addition, immunoblotting of AR was also performed on selected samples. Nuclear immunoreactivity of AR was found in seven of the nine urinary bladder cancers studied. AR showed variable staining intensity within a tumour. In the immunoblots, a 110 kDa AR signal was seen with anti-AR antibody, and faint bands of 90 and 60 kDa were also observed. Immunohistochemistry of p53 and c-erbB-2 was also carried out and compared with the distribution of AR. The high frequency of AR expression suggests a role for androgens in transitional cell carcinoma of human urinary bladder.     

Immunodetection of androgen receptor in human urinary bladder cancer

We investigated the expression of androgen receptor (AR) protein in transitional cell carcinoma of human urinary bladder in paraffin-embedded sections of tumours obtained from nine patients with urinary bladder cancer treated by radical cystectomy. In addition, immunoblotting of AR was also performed on selected samples. Nuclear immunoreactivity of AR was found in seven of the nine urinary bladder cancers studied. AR showed variable staining intensity within a tumour. In the immunoblots, a 110 kDa AR signal was seen with anti-AR antibody, and faint bands of 90 and 60 kDa were also observed. Immunohistochemistry of p53 and c-erbB-2 was also carried out and compared with the distribution of AR. The high frequency of AR expression suggests a role for androgens in transitional cell carcinoma of human urinary bladder.     

Feulgen-DNA-values in transitional cell carcinoma of the human urinary bladder.

Feulgen-stained imprint smears from 24 biopsy specimens from transitional cell carcinoma of the human urinary bladder were examined by means of scanning cytophotometry. One hundred randomly selected nuclei were measured from each biopsy specimen and the results compared with analogous measurements of nuclei from normal urinary transitional cell epithelium (13 cases in the control group). 97% of the nuclei in the control group were diploid. Well differentiated transitional cell carcinoma of the bladder mainly had a diploid DNA-stemline comprising 85% or more of the cells. One case of a well differentiated bladder cancer with a tetraploid DNA-stemline was found. The different cases of moderately differentiated bladder cancer showed diploid, triploid, tetraploid and hexaploid DNA-stemlines, while 4 out of 5 poorly differentiated tumours had a diploid DNA-stemline. The fifth extremely de-differentiated bladder carcinoma did not have any DNA-stemline at all. Together with these changes of the ploidy of the DNA-stemlines, increasing undifferentiation of the tumour tissue was combined with a reduced number of cells belonging to the tumour DNA-stemline, and increasingly scattered distribution of cells not contributing to the DNA-stemline. This indicates increased proliferative activity and/or chromosomal instability of the poorly differentiated cell population. Predominance of cells in the diploid, tetraploid and or octoploid intervals without marked frequency of DNA-values in the intermediary classes seemed to be a sign of clinical more "benign" bladder tumours, while a clinically more "malignant" tumour is characterized by increase of DNA-values in the triploid and hexaploid classes.     

Functional evidence of nitrergic neurotransmission in the human urinary bladder neck

Nitric oxide (NO) is involved in the non-adrenergic non-cholinergic (NANC) inhibitory neurotransmission of the lower urinary tract. However, functional evidence of this involvement in the human urinary bladder neck has not been consistently demonstrated. Therefore, the current study investigates the relaxations to endogenously released and/or exogenously added NO, in the human bladder neck. Urothelium-denuded bladder neck strips were dissected and mounted in isolated organ baths, containing a physiological saline solution (PSS) at 37 °C and continuously gassed with 5% CO₂ and 95% O₂, for isometric force recording. The relaxations to transmural nerve stimulation (EFS) or to exogenously applied NO, as an acidified solution of NaNO₂ were carried out on strips precontracted with phenylephrine, and treated with guanethidine and atropine, to block noradrenergic neurotransmission and muscarinic receptors, respectively. EFS (0.5–16 Hz) and exogenous NaNO₂ (1 μM to 1 mM) evoked frequency- and concentration-dependent relaxations, respectively. The nerve responses were abolished by the blockade of neuronal voltage-activated Na⁺ channels with tetrodotoxin, indicating their neurogenic character. N^G-nitro-l-arginine (l-NOARG), a NO synthase inhibitor, abolished the relaxations to nerve stimulation, which were partially reversed by the substrate of NO synthesis l-arginine. l-NOARG failed to modify the relaxations to exogenous NaNO₂. These results suggest that NO is the major NANC inhibitory neurotransmitter in the human urinary bladder neck. Blockers of NO synthase could be useful in therapy for the urinary incontinence produced by intrinsic sphincteric deficiency.     

Immunohistochemical demonstration of human chorionic gonadotropin in tumors of the urinary bladder

All transitional cell carcinomas of the bladder diagnosed in male patients within a five year period were studied for human chorionic gonadotropin (hCG) production. Biotin-avidin immunoperoxidase analysis for hCG was performed on all paraffin blocks containing carcinoma-in-situ, grade I, grade II, and grade III transitional cell carcinoma. Of a total of 29 patients, one case of carcinoma-in-situ (1/5), and five cases of grade III transitional cell carcinoma (5/15) were found to have hCG-positive tumor cells. The findings indicate that transitional cell carcinoma should be added to the list of malignant tumors capable of producing hCG.     

Expression of the TRPM8-immunoreactivity in dorsal root ganglion neurons innervating the rat urinary bladder

The neurochemical phenotypes of the transient receptor potential melastatin-8 (TRPM8)-immunoreactive afferent neurons innervating the rat urinary bladder were examined by using a highly sensitive tyramide signal amplification method, combined with wheat-germ agglutinin-horseradish peroxidase (WGA-HRP) retrograde tracing. TRPM8-immunoreactivity was detected in a small proportion of the WGA-HRP-labeled bladder afferent neurons in the dorsal root ganglia of the Th13-L1 (1.14%) and the L6-S1 (1.27%), and these neurons were small in size (<600 μm²). The 82.6 ± 3.8% of the TRPM8-immunoreactive bladder afferent neurons and 80.9 ± 1.5% of the total population of the TRPM8-immunoreactive afferent neurons in the observed dorsal root ganglia expressed NF200. On the other hand, the proportions of the co-expression of TRPM8 and nociceptive markers such as calcitonin gene-related peptide (CGRP), transient receptor potential vanilloid-1 (TRPV1), and isolectin B4 (IB4) in the bladder afferent neurons (81.5 ± 5.2% for CGRP, 36.1 ± 4.0% for TRPV1, and 15.8 ± 5.5% for IB4) were higher in comparison to those in the total population of the TRPM8-immunoreactive afferent neurons (21.9 ± 2.4% for CGRP, 16.6 ± 1.7% for TRPV1, and 5.4 ± 0.5% for IB4), although no significant difference existed for IB4. Our results suggest that the TRPM8-expressing bladder afferents should be classified as Aδ-fibers and C-fibers, while some of these afferents may be involved in nociceptive sensations.     

Distribution of NADPH-diaphorase and nitric oxide synthase-containing neurons in the intramural ganglia of guinea pig urinary bladder

The cell population and distribution of NADPH-diaphorase positive and NOS immunoreactive intramural ganglion cells were examined on stretched whole-mount preparations of the guinea pig urinary bladder which was divided into 3 regions: base, body and dome. The results showed that the highest frequency both of NADPH-d and NOS positive neurons was observed in the bladder base. Cell counts in the whole bladder showed that the number of NADPH-d positive neurons was much more than that of NOS immunoreactive neurons. Using neuron specific enolase (NSE) positive neurons as a reference (100%), NADPH-d positive neurons accounted for 84% while NOS immunoreactive neurons only made up 45% of the total neuronal population. These results, along with previous studies on the function of nitric oxide, suggest that nitric oxide may be involved in the relaxation activity in the bladder base during micturition. The significant difference in the number of NADPH-d positive and NOS immunoreactive neurons suggests that the localisation of one enzyme does not necessarily reflect the presence of the other.     

The prevalence and character of the muscularis mucosae of the human urinary bladder

We report the results of a histological evaluation of 335 consecutive autopsy specimens of apparently normal urinary bladder for the presence of a complete, partial or minimal muscularis mucosae. There were 164 females (49%) and 171 males (51%), ranging in age from 20 weeks of gestation to 102 years. A muscularis mucosae was present in 117 bladders (35%). The female to male ratio was 2:1, with 45% of female and 25% of male bladders containing a muscularis mucosae. The muscularis mucosae was complete in one case (1%), partial in 23 cases (6%) and minimal in 93 cases (28%). The occurrence of a partial or complete muscularis mucosae was ten times more likely in women than men. The presence of a muscularis mucosae in both women and men was not associated with any known disease process, nor was it related to patient age.