Evaluation of renal resistive index, maximum velocity, and mean arterial flow velocity in a hydronephrotic partially obstructed pig model.

RATIONALE AND OBJECTIVES: The authors investigate, in a controlled pig model, the usefulness of Doppler sonographic measurements of resistive index (RI), maximum velocity (Vmax), and mean velocity (Vmean) in diagnostic evaluation of the partially obstructed kidney. METHODS: Seven female pigs underwent surgical partial unilateral renal obstruction at the ureteropelvic junction. Doppler parameters of RI, Vmax, and Vmean were measured preoperatively and postoperatively in obstructed and contralateral nonobstructed kidneys. The General Linear Model multivariate analysis of variance was used for statistical analysis of data. RESULTS: There was no significant difference in the preoperative control values of RI (P = 0.71), Vmax (P = 0.27), and Vmean (P = 0.12). There was a statistically significant decrease in the Vmax and Vmean after postoperative day 5 in the obstructed kidney compared with the contralateral nonobstructed kidney (P = 0.01 and 0.03, respectively). There was no statistically significant difference in RI during the study. CONCLUSIONS: In this study, RI was not a reliable indicator of partial renal obstruction. Measurements of maximal and mean blood flow velocities reached discriminatory significance only after the fifth postobstructive day, proving insensitive in the immediate postobstructive period. Vmax and Vmean demonstrated a decrease in the partially obstructed renal collecting system after 5 days of obstruction and may serve as useful parameters in the evaluation of obstruction after the immediate acute period, when compared with a nonobstructed contralateral system.

The value of Doppler resistive index and peak systolic velocity in the evaluation of porcine renal obstruction.

PURPOSE: We evaluated the use of Doppler resistive indexes and systolic velocities as diagnostic tools in a partially obstructed animal model. MATERIALS AND METHODS: We studied 12 normal pigs by Doppler sonography, which served as control units. In 9 subjects partial ureteral obstruction was then created surgically, and 3 continued as controls. The presence or absence of obstruction was confirmed by ultrasound, antegrade nephrostography and intrapelvic pressure monitoring. Doppler parameters were measured 1 hour to 21 days postoperatively. Resistive index, and angle corrected peak and mean systolic velocities were measured bilaterally from samplings of interlobar and arcuate arteries. An average of 4 samplings per kidney was obtained at each session. Data from the control and obstruction groups were compared for statistical differences. RESULTS: In controls mean resistive index was 0.53, and peak and mean systolic velocities were 0.38 and 0.26 m. per second, respectively. In the subacute period after obstruction (defined as days 4 to 21) mean resistive index was 0.54, and peak and mean systolic velocities were 0.33 and 0.22 m. per second, respectively. Compared to control data there was no significant change in resistive index after obstruction (p = 0.6). However, peak and mean systolic velocities showed significant downward trends (p < 0.05). CONCLUSIONS: Known small sequential changes in renal blood flow after ureteral obstruction are reflected in flow velocities, as measured by duplex Doppler sonography. However, resistive indexes are not useful in assessing these changes in renal blood flow, and they do not correlate with the presence or absence of obstruction in this animal model.

A comparison of the high buoyant density proteoglycans isolated from the intervertebral discs of chondrodystrophoid and non-chondrodystrophoid dogs.

Groups of purebred beagles and greyhounds of similar ages (1.5-2.5 years) were used for the study. Intervertebral disc proteoglycans (PGs) were radiolabelled in vivo (with [35SO4(2-)], 24 hours and 60 days prior to euthanasia, when lumbar discs were dissected into nucleus pulposus (NP) and annulus fibrosus (AF). Aliquots of each disc region were separately analysed for total PG content as hexuronate. The remaining tissue was subjected to extraction with 4.0 M GuHCl. High buoyant density PGs were isolated from these extracts by CsCl density gradient ultracentrifugation. The hydrodynamic size and aggregatability of the 24-hour, 60-day-old, and resident PG populations were determined by Sepharose CL2B chromatography in the presence or absence of excess hyaluronic acid. While the hydrodynamic sizes of the newly synthesized (24-hour) disc PG preparations appeared to be similar, the 60-day-old greyhound disc PGs were found to be larger than the corresponding beagle disc PG populations. However, the keratan sulphate-core protein complexes prepared by chondroitinase ABC digestion of the newly synthesized (24 hour) disc PGs showed that the greyhound disc preparations were also larger than those from beagle discs. Approximately 80% of the newly synthesized PGs from beagle and greyhound discs were capable of aggregating with hyaluronic acid, however, this was reduced to 55% for the 60 day-old PGs in NP and AF and even less for the resident PG populations (as determined by hexuronate analysis). Significantly, PG aggregation was lower in the greyhound NP and AF preparations than in the corresponding PGs isolated from the beagle disc.(ABSTRACT TRUNCATED AT 250 WORDS)

The influence of scoliosis and ageing on proteoglycan heterogeneity in the human intervertebral disc.

Proteoglycans (PGs) purified from 19-year-old male (19M), 53-year-old female (53F), and 15-16-year-old adolescent idiopathic scoliotic (AIS) disc tissues were characterised by their aggregation properties with hyaluronic acid (HA), glycosaminoglycan (GAG), and amino acid composition, electrophoretic mobilities by composite agarose polyacrylamide gel electrophoresis (CAPGE), and hydrodynamic size by Sepharose CL2B gel permeation chromatography. Proteoglycan levels were similar in the AIS and 53F discs, but considerably higher levels were found in the 19M discs. Proteoglycan populations assessed by the aforementioned criteria demonstrated a similar level of heterogeneity in the AIS and 53F discs but this differed with the 19M sample. Two small Mr, low buoyant density PG species were particularly prominent in AIS and 53F discs: these were similar to two nonaggregating PG species formerly reported by di Fabio et al.; however, the corresponding PG species in 19M discs were of somewhat different composition. Collectively, these data suggest similar processing of PGs in the AIS and 53F discs. Furthermore, the pattern of normal age changes in the PGs from AIS discs appears to be disturbed, most likely arising as a consequence of the abnormal biomechanical forces intrinsic to the deformity.

Isolation and characterisation of a neutral proteinase from the canine intervertebral disc.

A neutral proteinase of 94 kDa capable of degrading gelatin, canine disc proteoglycan, and L-lysine and L-arginine peptide substrates has been isolated from the greyhound intervertebral disc. Strong inhibition of this proteinase with class-specific inhibitors, such as APMSF, TLCK and benzamidine indicated a 'serine'-type specificity. Metallo, aspartyl- and cysteine proteinase inhibitors were devoid of significant action. Degradation of the resident canine disc proteoglycan monomer by the disc proteinase was shown to occur at the hyaluronic acid binding region, thereby diminishing its ability to aggregate with hyaluronic acid. The hydrodynamic size of the proteoglycan degradation products was only slightly less than that of the intact disc proteoglycan subunits.

Cysteine proteinase inhibitors of the canine intervertebral disc.

Several species of cysteine proteinase inhibitors have been demonstrated in the greyhound intervertebral disc which were resolved into four species (Mr 15,800, 16,600, 17,200 and 17,800) by gelatin-SDS-polyacrylamide gel electrophoresis. Reductive alkylation did not affect their inhibitory capability nor their electrophoretic migration on gelatin-SDS-polyacrylamide gel electrophoresis. The cysteine proteinase inhibitors from the nucleus pulposus and annulus fibrosus were identical as assessed by the aforementioned criteria, although the level in the nucleus was found to be higher than that in the annulus. Ion-exchange chromatography demonstrated distinct acidic and basic forms of the disc cysteine proteinase inhibitor. The latter species was the most abundant and its Mr was determined to be 16,900 by gelatin-SDS-polyacrylamide gel electrophoresis. Both forms were shown to be strongly inhibitory against the cysteine proteinases, papain and ficin, but were less strongly inhibitory against cathepsin B (EC 3.4.22.1). Presumably these disc cysteine proteinase inhibitors play a regulatory role in the metabolism of proteoglycans and collagen by endogenous cysteine proteinases.

The response of the canine intervertebral disc to immobilization produced by spinal arthrodesis is dependent on constitutional factors.

Posterior lumbar spinal fusion was performed on five mature greyhounds. Two months prior to death, all of the surgical and five age-matched control greyhounds were given Na2(35)SO4 (1.0 mCi/kg) intravenously. All fusion animals were killed 6 months postoperation, and discs beneath the fusion mass as well as those adjacent to it (parafusion discs) were sampled separately and dissected into the nuclei pulposi and annuli fibrosi (AF). Proteoglycans (PGs) were extracted with 4.0 M GuHCl and then purified by CsCl density gradient ultracentrifugation. These PG monomers were subjected to Sepharose CL-2B chromatography, and their hydrodynamic size and ability to aggregate were determined. The level, extractability, and hydrodynamic size of PGs in the AF of fusion discs were found to be greater than those in control discs, as were the keratan sulfate core protein complexes prepared by chondroitin ABC lyase digestion. The ability of the 60-day-old PG subunit populations, isolated from fusion discs, to aggregate was also higher than controls. There was, however, no difference between the galactosamine/glucosamine, galactosamine/protein, glucosamine/protein, or hexuronate/protein ratios of PGs in fusion and control discs.

Variations of the proteoglycans of the canine intervertebral disc with ageing.

A group of young (2.0 +/- 0.6 years) (group 1) and old (9.7 +/- 1.5 years) (group 2) beagle dogs were given Na2 35SO4 (1.0 mCi/kg) intravenously 60 days prior to being killed to radiolabel their proteoglycans. Lumbar discs were removed and dissected into nucleus pulposus and annulus fibrosus. Proteoglycans were extracted at 4 degrees C from these tissues with buffered 4.0 M Gdn-HCl containing proteinase inhibitors, and purified by CsCl density gradient ultracentrifugation. The average hydrodynamic size and ability of the purified proteoglycans to aggregate in the presence of excess hyaluronic acid was determined by Sepharose CL-2B chromatography. The galactosamine/glucosamine ratios of these proteoglycans as well as their non-aggregating fractions were also ascertained. The proteoglycan content of discs of old animals was significantly less than in the young. The proportion of 35S-labelled, or non-labelled proteoglycans which could aggregate in the presence of hyaluronic acid was also much lower in the preparations isolated from the older discs. In contrast, the average hydrodynamic size of the non-aggregating proteoglycans isolated from the annuli fibrosi of group 2 animals were larger than the corresponding population of group 1 animals. Aminosugar analysis of these same proteoglycan fractions from older animals afforded galactosamine/glucosamine ratios (mean 1.81 +/- 0.14) which were less than the younger age group (mean 2.63 +/- 0.40). These data suggest that with ageing and degeneration the proteoglycans of the beagle disc undergo increased degradation with the accumulation in the annulus fibrosus of a population which is of larger average hydrodynamic size and richer in keratan sulphate than proteoglycans present in younger tissues.

The proteoglycans of the canine intervertebral disc.

The high-buoyant-density proteoglycans of the nucleus pulposus and annulus fibrosus of the beagle intervertebral disc have been isolated by CsCl density gradient ultracentrifugation. The sulphated proteoglycans were labelled in vivo with 35SO4, 24 h and 60 days prior to killing. The hydrodynamic size and aggregation of the 24 h, 60 day and resident (from hexuronic acid and hexosamine analysis) proteoglycan subunit populations were determined by Sepharose CL-2B chromatography in the presence or absence of excess hyaluronic acid. The hydrodynamic size of the keratan sulphate-proteoglycan core protein complexes were also determined by Sepharose CL-2B chromatography after chondroitinase ABC digestion of proteoglycans. When initially synthesised (24 h) or after 60 days, the percentage aggregation and hydrodynamic size of the proteoglycans derived from the annulus fibrosus were larger than those present in the nucleus pulposus. Hexosamine, hexuronic and protein determination of the high-buoyant-density fractions showed that the proteoglycans of the nucleus pulposus were richer in chondroitin sulphate than those in the annulus. However there was no difference in Mr of the chondroitin sulphate and keratan sulphate attached to the proteoglycans of the two disc regions, nor were differences detected by HPLC between the proportions of chondroitin 4-sulphate and chondroitin 6-sulphate present in these high-density fractions. In contrast, the low-buoyant-density (1.54 greater than p greater than 1.45) proteoglycan fractions and tissue residues remaining after 4 M GuHCl extraction were found to contain dermatan sulphate, suggesting the presence of a third proteoglycan species possibly associated with the collagen of the fibrocartilagenous matrix.

Effects of spinal fusion on the proteoglycans of the canine intervertebral disc.

Posterior lumbar two-level spinal fusion was undertaken in 10 mature beagles. The animals were sacrificed 6 and 12 months later. Two months before sacrifice control and experimental animals received intravenously Na2(35)SO4 (1 mCi/kg). Discs encompassed by the fusion and those adjacent to it were dissected into the nucleus pulposus and annulus fibrosus (AF), which were analysed separately. Proteoglycans (PGs) were extracted with 4.0 M guanidine HCl and purified by CsCl density gradient ultracentrifugation. The hydrodynamic size and ability of the PG subunits to aggregate in the presence of hyaluronic acid were investigated by Sepharose CL-2B chromatography. The PG subunits were analysed for their galactosamine (galN), glucosamine (glcN), hexuronic acid, and protein content or were subjected to digestion with papain or chondroitin-ABC-lyase to establish the size of the chondroitin (CS) and keratan (KS) sulphate chains and the KS-PG core protein complex. Decreased ability to aggregate of PGs isolated from discs 6 and 12 months after surgery was demonstrated. While their hydrodynamic size after 6 months was generally the same or smaller than those in control tissues, the PG population present after 12 months was larger, particularly in the AF. Analysis of PG subunits from fusion discs afforded galN/glcN, galN/protein, and hexuronic acid/protein ratios that were compatible with the presence in these tissues of PGs in which the proportion of CS attached to core protein was greater than in control tissues. These studies provide the first experimental evidence that a metabolic response of discs in a fused segment may be accompanied by the biosynthesis of a new PG population whose structure is similar to that present in immature tissues.