Stimulation of Structural and Functional Recovery of the Kidney in Rats with Postischemic Acute Renal Failure of Different Severity by Embryonic Protein-Peptide Complex Therapy.

In male rats, acute renal failure was simulated by clamping the vascular pedicle of the left kidney for 60 or 90 min and right-sided nephrectomy. In the control series, no therapy was performed. In the experimental series, the animals were daily injected subcutaneously with Cellex, a protein-peptide complex (PPC) chromatographically isolated from the brain tissue of pig embryos with a molecular weight of its components from 10 to 250 kDa. PPC was administered 5 times a week (10 injections) in a dose of 0.1 ml/kg (0.1 mg active substance per 1 kg body weight). Ischemia of a single kidney led to the development of acute renal failure, more severe after 90-min ischemia. PPC therapy reduced the severity of functional disorders mainly at the early stages (3 and 7 days) with normalization of blood concentrations of urea and creatinine, creatinine clearance, tubular reabsorption of sodium and calcium, including the cases with 90-min ischemia, which did not occur in the control series. PPC therapy also contributed to hypertrophy of many glomeruli, prevented the development of glomerulosclerosis, and reduced damage to the epithelium of the renal tubules. At the same time, neither pronounced lymphohistiocytic infiltration, nor focal nephrosclerosis typical of control series were observed.

Stimulation of Spermatogenesis and Synthesis of Testosterone by Allotransplantation of Neonatal Testicular Tissue under Tunica Albuginea of Cryptorchid Testis.

The abdominal type of cryptorchism was modeled on random-bred albino rats by replacing both testes from the scrotum into the abdominal cavity for 3 weeks; thereupon they were manipulated into the scrotum. In control rats, no additional surgery was performed. In experimental rats, the testicular tissue obtained from 1-2-day rat pups was transplanted under testicular tunica albuginea. Prior to orchiopexy, the weight of testes decreased by 62.5-64.1%. In 6 month after the surgery, it increased by 36.1% in the control group, whereas in experimental rats the weight of testes elevated by 123.2% and approximated the normal value. Histologically, the control group demonstrated persistent disturbance in spermatogenesis with emptiness of numerous seminiferous tubules where only Sertoli cells could be revealed and with pronounced dystrophic alterations in the spermatogenous epithelium of the partially preserved tubules where spermatogenesis was blocked at the spermatogonial level. In contrast, the transplantation region of the experimental testes exhibited formation of novel mature testicular tissue enclosed by a connective tissue capsule incorporating the seminiferous tubules with differentiated epithelium and with the clusters of Leydig cells in the stroma. In 6 month, spermatogenesis was observed in most seminiferous tubules of the host testicular tissue, which had spermatozoa in the lumens. To the moment of orchiopexy, the blood testosterone decreased by about 2.5-fold. In control group it remained diminished during entire observation period (up to 6 month), while in the experiment group its level normalized completely as early as in 2 month and remained even elevated to the end of observation period.

Pelvic Electric Potential as a Marker of Autonomic Dysfunctions and Risk Factor of Neurogenic Arrhythmias in Humans.

Differential high-resolution ECG (V1-V2) and pelvic electric potential measured between the coccyx and perineum were recorded simultaneously in resting supine position in men with autonomic nervous system disorders (N=37). In healthy volunteers (N=23), the effective (rms) value of PEP presented by median and interdecile range was 30 (20-80) µV within the frequency band of 0.03-80 Hz. In patients, the corresponding value was significantly higher: 140 (80-280) µV. In both groups, the amplitude harmonic spectrum of pelvic electric potential decreased monotonically with frequency according to 1/f1.6 law. In some patients (N=16), rare single or grouped high-amplitude impulses (up to 1 mV) of pelvic electric potential with total duration of about 1 sec were observed; of them, some persons (N=7) demonstrated practically one-to-one synchronous relations between these impulses and arrhythmia episodes indicating abnormal activity of the autonomic nervous system as their most probable common cause. The high-amplitude pelvic electric potential impulses were also observed in ECG records as interference signals with an amplitude attaining 50 µV. Thus, high-resolution ECG and pelvic electric potential can reveal the risk of abnormal neurogenic influences on the heart. The data obtained are discussed in relation to diagnostics of the autonomic nervous system disorders, neurogenic arrhythmias, and risk of sudden cardiac death.

Ectopic Organogenesis after Allotransplantation of Freshly Removed or Cryopreserved Neonatal Testicle under the Renal Capsule in Rats.

In experiments on white outbred male rats, a freshly removed (20 experiments) or cryopreserved (10 experiments) testicle from newborn rats (1-2 days after birth) was transplanted under the renal capsule after bilateral orchiectomy. In all experiments with transplantation of freshly removed testicle, it was engrafted. In 3 months, histological examination revealed the formation of mature seminiferous tubules, but spermatogenesis was blocked at the stage of spermatogonia; groups of proliferating Leydig cells in the loose connective tissue between the tubules were also seen. In 6 and 12 months, the status of the seminiferous tubules remained unchanged, but structures typical of the epididymis and developing vas deferens were revealed. The number of proliferating Leydig cells increased. The initially low testosterone concentration in the blood of castrated males increased significantly as soon as in 1 month after transplantation and grew up to 3 months, remaining at a level ~50% of normal. Engraftment of cryopreserved neonatal testicular tissue was observed in 60% cases, however, engrafted tissue, similar to the fresh one, retained the ability for organogenesis with the formation of mature seminiferous tubules, epididymis, and groups of proliferating Leydig cells. The dynamics of blood testosterone concentration in rats with cryopreserved and fresh transplantation was similar. Subcapsular transplantation did not adversely affect the kidneys, which was seen from normal histological structure of the kidneys and creatinine and urea concentrations in the blood.

Collagen-1 Membrane for Replacing the Bladder Wall.

We studied the possibility of using membrane fabricated from type 1 collagen isolated from cattle tissues (group 1) or porcine tissues (group 2) for replacement of the resected bladder wall defect in rabbits in order to retain functional volume of the organ. Satisfactory take of both types of collagen membranes with formation of competent anastomosis was observed. Histological studies revealed inflammatory process in the bladder wall at the site of contact with the implanted membrane (more pronounced in case of membranes from cattle tissues) that decreased by day 21 of the experiment. Bladder tissue ingrowth into the implant from was observed starting from day 14. The bladder capacity decreased in 7 days after surgery in both groups, presumably because of increasing tone of the organ wall resulting from surgical trauma and inflammation. In group 2, the bladder volume increased by day 14 after surgery and returned to normal by day 21, whereas in group 1 it remained below the control despite a trend to increase. These findings confirm good prospects of using collagen-1 membranes for plastic repair of the urinary bladder, the membranes from porcine collagen being more preferable.

Effect of Doxazosin on Autonomic Nervous Control and Urodynamics of Rat Urinary Bladder during Modeled Infravesical Obstruction.

The therapeutic effect of doxazosin (40 µg/kg/day over one month) on urinary bladder was examined in female rats with modeled chronic infravesical obstruction (IVO) produced by graduated mechanical constriction of the proximal urethral segment. In one month, IVO induced a pronounced vesical hypertrophy both in treated and untreated rats that manifested in increased bladder weight and capacity, the latter increment being pronouncedly greater in treated rats. In untreated IVO rats, infusion cystometry revealed elevated basal intravesical pressure of void bladder P0, markedly increased maximal (premicturitional) pressure Pmax, and increased amplitude of spontaneous oscillations of intravesical pressure ΔPdet in filled bladder. Doxazosin produced no significant effect on Pmax rise during IVO, but prevented elevation of P0 and increment of ΔPdet in filled bladder. During gradual filling of urinary bladder in control (intact) rats, the parasympathetic vesical influences increased progressively, while in untreated IVO rats, the adrenergic influences prevailed even at maximal filling of the bladder. In IVO rats, doxazosin prevented the bias of the sympathetic-parasympathetic balance in the filled bladder in favor of sympathetic influences, but did not prevent this bias in a void bladder. It is hypothesized that α-adrenoblockers improve micturition during IVO caused by benign prostatic hyperplasia not only by decreasing the urethral resistance to urine flow due to down-regulation of prostate smooth muscle tone, but also by a direct action of these blockers on detrusor adrenergic receptors and central structures involved in urinary bladder control.

Electrical Rhythms Revealed by Harmonic Analysis of a High-Resolution Cardiogram.

The front-end low-noise electronic amplifiers and high-throughput computing systems made it possible to record ECG with a high resolution in the low-frequency range including the respiration and Mayer frequencies and to analyze ECG with digital filtering technique and harmonic analysis. These tools yielded ECG spectra of narcotized rats, which contained the characteristic pulsatile triplets and pentaplets with splitting constant equal to respiration rate, as well as the peaks at respiration and Mayer frequencies. The harmonic analysis of ECG determined the frequency parameters employed to tune the software bandpass filters, which revealed the respiratory (R) and Mayer (M) waves in the time domain with the amplitudes of 20-30 µV amounting to 5% ECG amplitude. The depolarizing myorelaxant succinylcholine chloride capable to trigger various types of arrhythmias, transiently increased R-wave, inhibited M-wave, and provoked a negative U-wave within a heartbeat ECG cycle synchronously with inspiration. It is hypothesized that M-, R-, and U-waves in ECG reflect cardiotropic activity of autonomic nervous system. The respective spectral peaks in ECG can be employed to assess intensity of sympathetic and parasympathetic cardiotropic influences, their balance, and the risk of arrhythmias.

Ischemia in pelvic organs as an independent pathogenic factor in the development of benign prostatic hyperplasia and urinary bladder dysfunction.

Blood supply to the pelvic organs of outbred male rats was diminished by graduated constriction of the distal part of the inferior vena cava. Deficiency of intramural blood supply in prostate and urinary bladder was revealed by bioimpedance harmonic analysis according to the magnitude of first cardiac peak in the bioimpedance spectrogram. In 1-1.5 months, the histological examination revealed the glandular-stromal form of progressive benign prostatic hyperplasia in all ischemic rats. The development of hyperplasia was not accompanied by the changes in testosterone, dihydrotestosterone, or estradiol in blood and prostatic tissue. Assessment of vesical functional status by recording the intravesical pressure during infusion cystometry revealed an increase in the amplitude of spontaneous fluctuations of detrusor tone and intravesical pressure during bladder filling, which can be considered as indicator of detrusor hyperactivity. The data conclude that chronic ischemia of pelvic organs is an individual pathogenic factor in the development of benign prostatic hyperplasia and associated urinary disorders.

[Morphologic and functional heterogeneous changes of the urinary bladder different parts at rats with infravesical obstruction of the urinary tracts].

Infravesical obstruction of the lower urinary tracts of 30 rats was carried out by the measured constriction of the urethral prevesical parts. Morphologic and functional changes of the urinary bladder different parts were studied in 1 week and in 3 months. Compensatory hypertrophy of the detrusor was accompanied increasing of hypertrophied, atrophic and native forms of leiomyocytes, as so their transformation into myofibroblasts with connective tissue formation between the muscle fibers mainly in the neck of urinary bladder Contractility of the detrusor decreased with their tonus increasing mainly in the neck of urinary bladder too. Weakening effect of adrenalin in 3 month after obstruction significantly decreased, while in the body and neck of urinary bladder it virtually disappeared. Blockade of alpha-adrenoreceptors after noradrenalin stimulation resulted in enchancement of the hypertrophic detrusor contraction when tonus of the urinary neck decreased. On the contrary, in the intact urinary bladder the similarly influence resulted in weakening of contraction.

[Building of defects of the bladder wall by membrane, created on the basis of type I collagen (an experimental study)].

Experimental study was performed on 24 Chinchilla rabbits, which underwent resection of the bladder with building of defect by membrane "Collost", created on the basis of type I collagen. The functional state of the bladder in situ was assessed by infusion cystomanometry during repeated surgery at 7, 14 day and at 1, 3, 6 months, and at 1 and 1.5 years. It is found that developing detrusor pressure during bladder contractions was decreased by 10 times in the first week of the study; it was in line with the subcompensation after 3 months, and was restored at 6 month. At 1 and 1.5 years, increase of cumulative function of bladder while saving detrusor pressure was observed. Dilating cystoplastics using biopolymer "Collost" provides good long-term functional results.

[Biological aspects of choice of the intestinal segment for the cystoplasty].

Contractile activity of the iliac and sigmoid intestines versus detrusor activity, reabsorption and secretory activity of the iliac and sigmoid intestinal mucosa in contact with urine were studied in 30 rats. It was found that isolated segments of the iliac and sigmoid intestines have spontaneous contractile activity (stronger in the iliac intestine) while bladder segment contracted only in response to electric stimulation. A contraction-stimulating effect of acetylcholine and a relaxing effect of noradrenaline in experiments with the iliac intestine were close to their effects on the detrusor. The sigmoid intestine responded weaker to the above mediators. The iliac mucosa actively reabsorbed urinary urea, creatinin, glucose causing elevation of their concentrations in blood as well as K, Na, Ca, CI, P and secreted protein in urine leading to hypoproteinemia. The sigmoid mucosa showed weaker metabolic activity. The results of the study demonstrate importance of consideration of biological properties of different intestinal regions for choice of a cystoplasty method after cystectomy.

[Mechanism of action and efficacy of litium chloride in thermal ischemia of the kidney].

A protective effect of lithium chloride (LC) in thermal ischemia of the kidney of different duration was studied in rats. LC efficacy was estimated by functional activity of ischemic kidney in early and late reperfusion period, by damage to mitochondria of tubular epithelial cells and production of active oxygen forms (AOF) and nitric oxide (NO). LC has a marked anti-ischemic effect. In thermal renal ischemia for 40 and 60 min LC provides functional safety of the ischemic organ. In longer ischemia, 50% of the animals died. The mechanism of the protective action of LC is related to reduction of APK production, support of a high transmembrane potential of mitochondria and NO synthesis redistribution in the cells of tubular epithelium cells.

Heterogeneity of morphological and functional changes in various compartments of rat urinary bladder in infravesical obstruction of the urinary tract.

Infravesical obstruction of the lower urinary tract was simulated in rats by dosed constriction of the prevesical portion of the urethra. The functional and morphological changes in various urinary bladder compartments were evaluated after 1 week and 3 months. The development of compensatory hypertrophy of the detrusor was associated with an increase in the number of hypertrophic, atrophic, and young leiomyocyte forms and their transformation into myofibroblasts, with the formation of connective tissue laminae between myofibril bundles mainly in the zone of urinary urinary bladder neck. Specific contractility of the detrusor strips decreased with increasing their tone, which was most pronounced in the neck zone. The relaxing effect of norepinephrine was significantly lower after 3 months of obstruction and virtually disappeared in the zone of the urinary bladder body and neck. Blockade of a-adrenoceptors after adrenostimulation with norepinephrine stimulated contractions of the hypertrophic detrusor against the background of reduced tone of the urinary bladder neck, in contrast to intact urinary bladder where this treatment reduced contractions.

Morphological changes in the kidneys of rats with postischemic acute renal failure after intrarenal administration of fetal mesenchymal stem cells from human bone marrow.

Chronic experiments on outbred albino rats were performed to compare the dynamics of histological signs for postischemic renal injury (90-min thermal ischemia) after intraparenchymal injection of cultured fetal MSC from human bone marrow. Functional indexes of the ischemic kidney were predetermined. In the early period after ischemia (day 4), administration of human bone marrow MSC was followed by the increase in blood flow in the microcirculatory bed and decrease in the degree of alteration in renal tubules. An increase in the area of zones with histological signs for normal function of tubules was accompanied by the improvement of biochemical indexes for renal function. In the delayed period, a protective effect of cell therapy was manifested in the prevention of death of renal tubules. Mild calcification of the necrotic tubular epithelium served as a marker of this process. Human bone marrow MSC were labeled with the fluorescent probe Calcein. These cells migrated from the site of injection, spread in the interstitium, and retained viability for 7 days. During this period, some cells were incorporated into the lumen of renal tubules.

[Function of the urinary bladder detrusor after injection of botulinic toxin].

The effect of botulinic toxin of type A (BoNT) on urinary bladder (UB) function was studied during operation in situ in healthy rats using the method of infusion cystomanometry after a single injection of the toxin into the UB wall 3 to 30 days after the injection. Contractility of the muscular fragments in vitro was assessed in the isometric regimen in electric stimulation, depolarization of cellular membranes by ion [K+] excess, addition of noradrenalin (10(-5) M). In response to botulinic toxin injection 47-64% rats vs control animals (23%) operated on without toxin had no phasic UB contractions, detrusor pressure (Pdet) after the infusion was higher which indicated retension. In rats with intact UB contractility, contractions were stronger (Pdet 24-34 cm H2O), but rare (3-5/min) vs controls (19 cm H2O, 5/min, respectively). Contractility in vitro in response to electric stimulation regressed by 60-85% after toxin infusion. Contractility in solution [K+] regressed less (by 65, 36, 16% in 3, 10 and 30 days, respectively). No significant changes of noradrenalin-induced reactions were found. The results illustrate possible direct myogenic effect of the toxin on detrusor muscle.

[Regeneration of testicular tissue and restoration of rat fertility in xenotransplantation of enriched fetal cell cultures in bilateral abdominal cryptorchism].

The study of cell cultures enriched with stem and progenitor cells in the treatment of experimental hypergonadotropic hypogonadism was made on 30 white non-inbred rats with experimental cryptorchism who have undergone xenotransplantation of human fetal enriched cell cultures. Spermatogenic epithelium on histological sections was studied on day 14 and 28 after xenotransplantation with calculation of the spermatogenesis index. The fertility index was estimated for each of the groups. Transplantation of enriched cell cultures enhances efficacy of restoration of adequate germinogenic and spermatogenic testicular function, accelerates recovery of spermatogenesis and fertility with high indices of spermatogenesis and fertility but effect of the above recovery after treatment with enriched cell cultures can be seen not earlier than 72 days after transplantation.

Effect of xenotransplantation of cell cultures enriched with stem and progenitor cells on hormonal profile of rats with abdominal cryptorchism.

We present the results of application of cell cultures enriched with stem and progenitor cells for the treatment of experimental abdominal cryptorchism in outbred albino rats. Xenotransplantation of human fetal enriched cell cultures was performed to animals with experimental cryptorchism during orchiolysis. Total testosterone, luteinizing and follicle-stimulating hormones were assayed by immunochemiluminescent method. It was found that xenotransplantation of cell cultures enriched with stem and progenitor cells normalized the level of total testosterone, decreased the concentrations of gonadotropic hormones, reduced hyperplasia of Leydig cells and the number of chromaffin granules, and restored normochromism of Leydig cells nuclei.

Effects of ischemic and hypoxic preconditioning on the state of mitochondria and function of ischemic kidneys.

Laser confocal microscopy showed that fluorescence of tetramethylrhodamine ethyl ether probe specifically accumulating in energized mitochondria significantly decreased in renal tubular epithelium after 40-min thermal ischemia, while fluorescence of dichlorodihydrofluorescein and diaminofluorescein probes in the same structures increased under these conditions, which attests to increased generation of ROS and NO, respectively. These forms were generated predominantly in mitochondria of tubular epitheliocytes. Hypoxic preconditioning (a series of sessions of breathing hypoxic mixture) preserved functional activity of mitochondria and prevented activation of ROS and NO generation. Ischemic preconditioning of the kidney consisting of three preliminary episodes of vascular clamping (5 min with 5 min reperfusion periods) also increased the percentage of functionally active mitochondria and prevented activation of NO synthesis without appreciably modifying ROS production. Both protective methods significantly reduced the severity of postischemic dysfunction of the kidney.

Experimental intravenous cell therapy of acute and chronic renal failure.

The therapeutic effect of intravenous injection of human fetal bone marrow mesenchymal stem cells or summary culture of kidney cells were studied on models of chronic or acute renal failure in outbred albino rats. Both cell types promoted improvement and normalization of the renal function in rats with stable chronic renal insufficiency (2 weeks after kidney cell injection, 1 month after bone marrow mesenchymal stem cell injection). Renal function remained normal or subnormal during the delayed period (3-3.5 months after injection). In rats with latent stage of chronic renal insufficiency, exacerbation was induced by additional 40-min ischemia. All rats receiving intravenous injection of saline died. Improvement of the functional parameters started 2 weeks after injection of kidney cells or bone marrow mesenchymal stem cells, and normalization was observed after 1.1-5 months. During the delayed period (after 3-4 months), functional parameters retained at normal or subnormal levels. In experimental series III, all rats with acute renal failure intravenously injected with saline (control) died from uremia on days 2-4. After injection of kidney cells 50% rats survived and renal function in these animals returned to normal after 2 weeks. After injection of bone marrow mesenchymal stem cells 83% rats survived, functional parameters returned to normal after 3 weeks.