Surgical Management of Obstructive Urolithiasis and Fourier Transform Infrared (FT-IR) Spectroscopic Identification of Uroliths in Dogs: A Report of Six Cases


 Six dogs with signs of obstructive urolithiasis presented to University Veterinary Hospitals Mannuthy and Kokkalai, Kerala Veterinary and Animal Sciences University over a period of two months formed subject for the present study. All the animals were having history of difficult urination with clinical signs like stranguria, pollakuria, anuria and/or haematuria. Haemato-biochemical investigations, blood gas analysis, urinalysis, survey radiographs and ultrasonography were performed to arrive at a conclusive diagnosis. Surgical interventions were done under general anaesthesia using either urethrotomy, cystotomy or a combination of both, depending upon the site of obstruction. Post-operative pH modulators, anti-spasmodics, antibiotics and analgesic therapy were employed depending upon the condition of patient and all the animals had an uneventful recovery. When stones retrieved through surgery were subjected to Fourier Transform Infrared Spectroscopy (FT-IR) to identify the chemical composition, three samples were identified as a combination of struvite, calcium oxalate dihydrate and calcium apatite crystals and three samples were a mixture of calcium oxalate monohydrate and calcium phosphate. Identification of specific calculi composition using spectroscopy was helpful in prevention of recurrence through management practices and therapeutic diet postoperative.


Introduction
Urolithiasis is a general term referring to the causes and effects of stones anywhere in the urinary tract, causing urinary tract disease demanding rapid de nitive diagnosis for immediate surgical and/or medical interventions (Osborne et al. 1986). The precipitation-crystallization theory of urolithiasis explains that supersaturation of urine with ions resuts in precipitation and formation of particles, that grow into small crystals and aggregates into larger crystals and ultimately results in calculi formation if the microenvironment is favourable (Dibartola et al. 1981). The factors in uencing development of canine urolithiasis and its characteristics are age, sex, etiological factors such as metabolic disorders, ionic transport within the intestinal tract and in the kidney, diet including uid intake, geographical location and climate (Osborne et al. 2009). De nitive theraputic management of urolithiasis should be directed at early con rmation and effective interventions including, surgical therapy, post-operative medications, medical dissolution and urinary pH modulation in conjunction with dietary modi cations and theraputic diets (Koehler et al. 2008) The understanding of the chemical composition of urinary stones is of interest for proper identi cation of the type of stone, to provide clues to etiopathogeny and prevention of recurrence. Fourier Transform Infrared Spectroscopy (FT-IR) is based on unique wave patterns generated when infrared waves encounter a sample, where some waves get absorbed by the sample (absorbance) and some waves pass through the sample (transmittance). The resulting spectrum is a molecular nger print of the sample which can be compared with a known reference spectra for identi cation (Koehler et al. 2008). Though numerous studies in the past focused on FT-IR Spectroscopic characterization of kidney stone compositions in clinical urology, there are only limited literature on FT-IR quanti cation of uroliths in small animals. By virtue of its accuracy and expeditiousness, this method proves to be a valuable technique for the analysis of urinary calculi composition, applicable in veterinary urology.
The present study was conducted in six dogs presented with signs of obstructive urolithiasis varying in the sites of obstruction. Haematobiochemical, blood gas analysis and radiographic examinations were conducted in all the cases and surgical management was done using either urethrotomy, cystotomy or a combination of both. Stones thus collected were subjected to FT-IR Spectroscopy, a speci c, rapid and versatile analytical technique which provides quantitative as well as semi-quantitative analysis of urinary stone composition.

Materials And Methods
Six dogs with signs of obstuctive urolithiasis presented to university veterinary hospitals, Mannuty and Kokkalai, Kerala Veterinary and Animal Sciences, University, India over a period of 2 months (November and December, 2019) formed the subjects for the present study. Age, breed, sex and body weight of the animals were recorded at the time of presentation. Detailed anamnesis including duration of illness, severity of the condition, whether the obstruction was complete or partial, previous ailments and medications if any and clinical symptoms including anorexia, lethargy, depression, vomiting, crouching stance, abdominal distention, stranguria, pollakuria, dysurea and haematuria. Thorough physical examination of the patients were carried out to record the Respiratory rate (RR), Pulse rate (PR), rectal temperature, colour of mucous membrane, hydration status, abdominal palpation and appearance of prepuce and penile region. Complete blood count, serum biochemical analysis and blood gas analysis were conducted along with right lateral survey abdominal radiographs ( Fig.1) to visualize the site of obstruction and ultrasonography to evaluate the extent of bladder distension/rupture/cystitis. An ECG was recorded to monitor the patient for the presence of hyperkalemia induced cardiac dysarrhythmia. Aseptically collected urine samples were subjected to urinalysis to determine the pH value and centrifuged to obtain the sediments for microscopical analysis.

Surgical Technique
Cystotomy, urethrotomy or combination of both were the surgical options depending on the site of calculi once the retrograde urohydropropulsion failed. All the animals were pre-medicated with atropine sulfate at the dose rate of 0.045 mg/kg bodyweight followed by xylazine hydrochloride at 1.0 mg/kg bodyweight intramuscularly. Anaesthesia was induced with intramuscular injection of ketamine hydrochloride and midazolam (1mg/ml) at the dose rate of 5.0 mg/kg body weight and 0.05 mg/kg bodyweight respectively and anaesthesia was maintained with two percent iso urane. Cystotomy was performed though caudal midline laparotomy in the female animal (Case I) to remove the single large cystolith which was constantly irritating the bladder mucosa.
Penile urethrotomy was performed in cases III, IV and VI to remove uroliths present behind the os penis, followed by urethrostomy (Fig. 2a). A combination of cystotomy and urethrotomy were performed in case II to remove multiple number of stones present throghout the course of urethra and in the bladder. There was cystorrhexis in case V along with focal necrosis of bladder wall and mucosal hypertrophy (Fig. 2b) which was managed by partial cystectomy. The uroliths retrieved through surgery are depicted in the Fig.3.
Intraoperatively electrolyte imbalance was corrected based on blood gas analysis with 0.9 percent normal saline, 25 percent dextrose and 7.5 percent sodium bicarbonate injections. Post operatively the animals were given amoxycillin clavulanate at the dose rate of 20 mg/kg for ten days, meloxicam at the dose rate of 0.2 mg/kg body weight as analgesic and cyclopam 10 mg total dose as antispasmodic orally for four days. Post operative dietary management and urinary pH modulators were adviced on a case to case basis depending on the speci c urolith composition and urine pH. In cases having abnormally acidic urine, disodium hydrogen citrate was adviced for urine pH correction and DL-methionine and ascorbic acid were adviced for acidi cation of urine in animals with an increased urine alkalinity usually accompanied by urinary tract infections.

Fourier Transform Infrared Spectroscopic analysis of uroliths
The speci c chemical composition of stones retrieved surgically were analyzed quantitatively using FT-IR Spectroscopy. The samples were ground to ne homogenous powder, mixed with Potassium Bromide (KBr), an inert carrier which has no absorption in the spectral region to be investigated. It was then pressed in to a pellet using high pressure in a hydraulic pellet press, loaded in the IR sample holder and spectroscopy was performed using FT-IR spectrometer to obtain the characteristic scan pro le.

Results
The breed, sex, age, body weight and physiological parameters of the animals under study are enlisted in Table 1. The average age of animals presented was 8.5 years and ve out of six animals were male dogs. All the animals had history of not passing urine for past one to two days except the female which had history of haematuria for past 3 months. The severity of clinical signs depended on the duration of obstruction and whether the obstruction was complete or partial. The animals exhibited anorexia, lethargy, vomiting crouching stance and one animal had cyanotic and in ammed penis. Five out of six dogs had multiple uroliths and radiography con rmed the site of obstruction as behind the os penis in four cases, thoughout the course of urethra and in the urinary bladder in one case and a single large cystolith in the female dog (Fig. 1). Ultrasonography revealed intact bladder in all cases except one with free uid in the abdomen suggestive of bladder rupture. The mean haematological parameters are represented in Table 2, which were within the normal range except for mild neutrophilic leukocytosis and anaemia in three out of six cases. Serum biochemical analysis revealed elevated blood urea nitrogen and creatinine values indicating post renal azotemia in cases with complete urinary obstruction. Hyperkalemia and primary metabolic acidosis were typical of obstructive urolithiasis in blood gas analysis in four out of six cases (Table 3). An electrocardiogram also revealed increased amplitude of T wave suggestive of hyperkalemia in cases with obstructed out ow. Urinalysis showed alkaline pH of 7.5 to 8.0 and microscopic analysis of urine sediments revealed struvite crystals in rst three cases where as pH was towards acidic side (6.0 to 6.5) in other cases which showed presence of calcium oxalate monohydrate crystals on microscopic analysis (Table 1).   and apatite crystals. The absorbance of calculi with their wave number and percentage absorption are enlisted in Table 4 Table 5 and the FT-IR spectrum of the calculi is depicted in Fig. 5. The grouping of 5 bands in a staircase pattern between 3477 and 3047 cm combined with other bands at 2291, 1620, 1320, 885, 662, 781 and 517 cm − 1 con rmed the presence of calcium oxalate monohydrate crystals (COM). The presence of calcium phosphate crystals was con rmed by detecting absorbance at 1040 cm − 1 specici c to phosphorous.  Discussion Urolithiasis is considered a surgical emergency as they may complicate with cystorrhexis and uroabdomen when urine is retained for more than 32 to 48 hours. Clinical cases of urolithiasis are mostly recorded in male animals and the incidence is more in older animals compared to younger age groups (Fromsa speci cally focus the site of obstruction as the stones were radio opaque in nature except cystiene and urate ). Urine pH was consistently towards alkaline side when the major stone composition was struvite but was acidic in COM and calcium phosphate urolithiasis (Palma et al. 2013).
Treatment was directed to immediately relieve the obstruction, correction of post-renal azotemia, hyperkalemia, acid-base imbalance and dehydration.
Alleviation of primary causes of the disease by dietary modi cation, medical management and uid therapy were also followed. Cystotomy, urethrotomy or combination of both were the surgical options depending on the site of calculi once the retro grade urohydropropulsion failed. In the present study, three out of six cases were managed with urethrotomy alone, one with cystotomy and two cases by urethrotomy combined with cystotomy.  Rest of the three samples were identi ed as mixture of COM and calcium phosphate. The important spectral characteristics of COM was grouping of ve bands between 3477 and 3047cm − 1 , due to symmetric and asymmetric O-H stretch (Kanchana et al. 2009).The staircase pattern of ve absorption bands between 3477 and 3047cm − 1 in the present study con rmed the presence of COM crystals ( Table 5). The absorptions at 2291cm − 1 and 885cm − 1 was due to C-Cstretching mode, two bands at 662 and 781cm − 1 were due to the out of plane O-H bending and C-CH bending mode respectively and absorption at 517cm − 1 arised due to O-C-O in plane bending (Sundaramoorthi et al. 2007). The absorption band at 1620 cm − 1 and 1320 cm − 1 was due to the vibration of C = O and C-O respectively (Tsay 1982). Hence, in addition to the grouping of 5 bands, the presence of COM was con rmed with the presence of absorption bands at all the above speci ed wave numbers for COM. The discrete formation of two bands at 780 and 517cm − 1 was important in differentiating COM from COD FT-IR spectrum of urinary stone containing magnesium ammonium phosphate hexahydrate (Struvite), calcium oxalate dihydrate and apatite crystals Figure 5 FTIR spectrum of urinary stone containing calcium oxalate monohydrate (COM) and calcium phosphate