Effect of therapeutic ultrasound and diathermy on oxidant–antioxidant balance in dogs suffering from hind quarter weakness

Abstract

The present study was conducted to evaluate the erythrocytic oxidant–antioxidant balance in dogs suffering from hind quarter weakness (HQW) and treated with conventional drug therapy (CDT, n=8) alone and in combination with therapeutic ultrasound (US, n=8) and shortwave diathermy (SWD, n=8). A further eight healthy dogs were used as controls. For oxidant level, Lipid Peroxidation (LPO) and antioxidant levels Reduced Glutathione (GSH), Superoxide Dismutase (SOD) and Catalase (CAT) were evaluated in erythrocyte before (day 0) and after 3, 7, 14 and 28 days of therapy. In dogs with HQW, LPO in terms of Malondialdehyde (MDA) production, was found significantly (P<0.05) increased compared to a healthy control on day 0. Thereafter, a significant (P<0.05) decrease in the level of LPO was noticed. Amongst antioxidant enzymes, activities of GSH and CAT decreased significantly (P<0.05) whereas, the level of SOD increased significantly (P<0.05) until day 28 post treatment. Studies revealed that SWD and therapeutic ultrasound in conjunction with CDT can be used to counter free radical-mediated oxidative cell injury, induced by HQW in dogs. However, SWD proved to be better in minimizing excess free radicals production and activating antioxidant defense system as compared to therapeutic ultrasound.

Keywords:

• dogs
• oxidant–antioxidant
• oxidative stress
• ultrasound
• diathermy
• hind quarter weakness

Introduction

Painful and/or debilitating neurological disorders primarily involving the vertebral column and spinal cord are commonly encountered in small animal practices, especially in dogs. Animals with spinal disorders are presented with focal or generalized pain, varying degrees of paresis, paralysis and inability to urinate (Nelson and Couto 2004). The most frequent manifestation of spinal cord affections is Hind Quarter Weakness (HQW). It is the loss of bilateral motor function of the rear limbs due to dysfunction of neural or muscular system.

Current management of acute spinal cord injury in small animals involves diagnosing and relieving gross misalignments and other structural problems of the spine, minimizing cellular-level damage and stabilizing the vertebrae to prevent further injury. Once a patient is stabilized, supportive care and rehabilitation strategies play an important role in the functional regeneration of the spinal cord (Ettinger and Feldman 2010). Physiotherapy is the use of non-invasive techniques that act by decreasing pain, inflammation and swelling, improving blood supply, minimizing muscle atrophy and thus promoting early recovery and back-to-normal or near-normal function (APTA 2008). Physiotherapy is in fact complementary to conventional treatment and best used in collaboration with it (McGowan et al. 2007). Conventional treatment by corticosteroids, non-steroidal anti-inflammatory drugs (NSAIDs) and nervine tonics yielded only limited success in cases of HQW in dogs. However, when combined with other physiotherapeutic modalities viz. acupuncture, ultrasound and interferential, a higher success rate was noticed (Sharma 2005; Maiti et al. 2007).

Normal metabolic processes produce free radicals continuously, but their rate of production increases during certain inflammatory or other disease conditions (Bernabucci et al. 2005). Oxidative stress results when there is an imbalance between the generating and scavenging activity of radicals, resulting in oxidative products and tissue damage (Saleh et al. 2011). When free radical generation overpowers the antioxidant defense, free radicals can interact with endogenous macromolecules leading to metabolic dysfunction and bimolecular oxidative damage (e.g. damage to lipids, DNA, carbohydrates and proteins), which contribute to pathological changes in the tissues (Trouba et al. 2002; Valko et al. 2007). Estimation of antioxidant enzymes activities viz. SOD, CAT and GSH and level of oxidant (LPO) in blood are indirect but reliable methods for assessment of free radical activity and oxidative stress (Fang et al. 2002).

Ultrasound and Shortwave Diathermy (SWD) have been found to be beneficial in the treatment and management of various degree of spinal trauma, pain, HQW and posterior paresis in dogs. However, their oxidant–antioxidant response has not been documented. Thus, the present work was aimed at investigating the ameliorative effect of ultrasound and SWD therapy against oxidant/antioxidant imbalance in dogs affected with clinical HQW disorder.

Materials and methods

Selection of animals and treatment protocol

A total of 24 dogs (2.5 months–13 years of age) of either sex suffering from HQW presented to Referral Veterinary Polyclinic of Indian Veterinary Research Institute were included for this study. All of the animals were randomly divided in three equal groups (II, III and IV) of eight dogs each. Another set of eight age-matched healthy dogs constituted the healthy controls group (I). Dogs in group II were treated with conventional drug therapy (CDT) alone. In addition to the CDT, animals of group III and group IV were also treated with ultrasound and SWD, respectively. CDT was administered for 14 days using methyl prednisolone acetate (Depomedrol, Pfizer Product India Pvt Ltd., Mumbai-400102) @ 30 mg/kg body weight intramuscular on day 1 and later on 15 mg/kg body weight I/M. on alternate days, Meloxicam (Melonex, Intas pharma, 6th Floor, Chinubhai Center, Ashram Road, Gujarat, 380009) @ 0.2 mg/kg body weight I/M daily, Gabapentine and Mecobalamine (Neurokind-G, Mankind Pharma Ltd., Okhla, New Delhi 110023, India) tablet, orally once daily and Vitamin B1, B6, B12 and Panthenol (Cyanocal-16, Ozone Pharmaceutical, New Delhi 110058, India), 2 ml, I/M on alternate days. Ultrasound therapy was applied after clipping hairs and applying ultrasound jelly to ensure perfect contact between transducer head and skin on the affected area. Sonication was performed at a frequency of 1 MHz, an intensity of 0.5 watt/cm² (SATA) for 5 minutes/day for 14 days in a pulsed mode (1:4). The transducer head of the ultrasound machine was moved slowly in a proximal–distal linear fashion on the skin surface. SWD therapy (200 mA intensity and 9 volts) using two pads at lumber region, was performed daily for 10 minutes for 14 days.

Blood collection and processing for assays of oxidant/antioxidant balance

Three ml each of venous blood samples were collected from each animal on days 0, 3, 7, 14 and 28 in two disposable plastic syringes containing heparin (20 IU/ml). Blood samples were centrifuged at 2000 rpm for 10 minutes and used to estimate the oxidant–antioxidant balance parameters. One blood sample was used to prepare hemolysate and the other for RBC suspension. To prepare hemolysate, erythrocytes were washed three times with normal saline solution and finally 10% hemolysate was prepared by adding chilled distilled water. RBC suspension was prepared by adding an equal volume of the erythrocytes and normal saline solution. Hemolysate and RBC suspension were kept at −70°C and used for oxidant–antioxidant assay within 6 hours. From hemolysate, SOD activity (Marklund and Marklund 1974), CAT activity (Bergmayer 1983) and malonaldialdehyde (MDA) concentration (Placer et al. 1966) were estimated. MDA is a reliable marker of LPO. The concentration of GSH in RBC suspension was estimated using the method of Prins and Loos (1969).

Statistical analysis

The values were expressed as mean±SE. Statistical analysis was carried out by one way Analysis of Variance (ANOVA) for group, time and their interaction effects and followed by Duncan's multiple range tests using Statistical Package for Social Sciences software (SPSS 16.0, Chicago, IL, USA). The level of statistical significance for all comparisons was established at P < 0.05.

Results and discussion

The effects of different treatments on oxidant–antioxidant parameters are shown in Tables 1–4. On day 0, the LPO in terms of MDA production was significantly (P < 0.05) higher in all dogs with HQW compared to the healthy control. Thereafter, LPO exhibited a continuous significant (P < 0.05) decrease at all intervals in all the treated groups. The decrease was highest in group IV followed by groups III and II. The values differed significantly amongst all the groups at corresponding intervals, with the exception of day 14 where the LPO level differed non-significantly between groups II and III (Table 1). In the present study, the oxidant/antioxidant balance shifted towards oxidative stress in all dogs suffering from HQW, as evidenced by an increase in the levels of LPO in terms of MDA. Normal metabolic processes produce free radicals continuously, but their rate of production increases during certain inflammatory or other disease conditions (Bernabucci et al. 2005). Under normal conditions, free radicals are neutralized by efficient antioxidant systems (Nockels 1996). Higher LPO levels are suggestive of enhanced oxidative damage to erythrocytes, either due to excessive production of free radicals or compromised/exhausted antioxidant defense in the affected animals. MDA is a breakdown product that is frequently quantified as a measure of lipid hydro peroxides. MDA assay has been found to be one of the better predictors of oxidative damage and often shows excellent correlation with other markers, such as isoprostanes, which are considered to be the most reliable markers of LPO (Morrow 2000). Similar findings of increased levels of LPO were reported in various disorders like inflammation (Lykkesfeldt 2002), canine demodicosis (Dimri et al. 2008a), caprine scabies (De and Dey 2010), sarcoptes in dogs (Camkerten et al. 2009), buffalo (Dimri et al. 2008b) and camel (Saleh et al. 2011). After treatment began, a continuous significant (P < 0.05) decrease in LPO in all of the treated groups suggested a decrease in excess free radicals. The biggest decrease in LPO in group IV followed by groups III and II indicated that the least oxidative damage to erythrocytes occurred when SWD was used as an adjunct therapy to conventional treatment, as compared to therapeutic ultrasound.

Table 1. Effect of different treatments on LPO (nmol MDA/g Hb).

Group	Day 0	Day 3	Day 7	Day 14	Day 28
I	1.29^aD ±0.009	1.28^aC±0.007	1.29^aC±0.009	1.24^aA±0.006	1.26^aB±0.007
II	3.63^bE±0.015	3.44^dD±0.031	3.04^dC ±0.559	2.03^cB±0.113	1.77^dA±0.047
III	3.64^bE±0.017	3.31^cD±0.009	2.94^cC ±0.027	1.99^cB ±0.092	1.68^cA±0.007
IV	3.63^bE±0.014	3.25^bD±0.010	2.90^bC ±0.009	1.68^bB ±0.008	1.65^bA±0.007
^A–EValues in the same row without a common superscript letter are significantly different (P<0.05).
^a–dValues in the same column without a common superscript letter are significantly different (P<0.05)

Table 2. Effect of different treatments on SOD (U/mg Hb).

Group	Day 0	Day 3	Day 7	Day 14	Day 28
I	0.32^aA±0.028	0.33^aA±0.025	0.34^aAB±0.017	0.36^aB ±0.023	0.35^aB±0.017
II	0.91^bE±0.023	0.86^bD±0.037	0.79^bC±0.031	0.52^dB±0.008	0.44^dA±0.008
III	0.88^bE±0.051	0.84^bD±0.045	0.80^bC±0.051	0.50^cB±0.009	0.41^cA±0.010
IV	0.91^bE±0.045	0.83^bD±0.035	0.78^bC±0.037	0.48^bB±0.016	0.40^bA±0.010
^A–EValues in the same row without a common superscript letter are significantly different (P<0.05).
^a–dValues in the same column without a common superscript letter are significantly different (P<0.05).

SOD level was significantly (P<0.05) higher in dogs with HQW as compared to the healthy control on day 0. Thereafter, SOD exhibited a continuous significant (P < 0.05) decrease at all intervals in all the treated groups. The decrease was greatest in group IV followed by groups III and II. There was a significant (P<0.05) difference in the SOD values amongst the treated groups on days 14 and 28. It is well established that when the risk of oxidative damage increases, endogenous antioxidant protection also increases (Basha and Rani 2003). Various kinds of stressors increase LPO levels and therefore SOD activity (Lata et al. 2004). SOD is a natural antioxidant of the body. SOD accelerates the dismutation of superoxide radicals () to hydrogen peroxide (H₂O₂), whereas CAT catalyzes the breakdown of toxic H₂O₂ produced in the cell to O₂ and H₂O (Linares et al. 2007). The present increase in SOD activity in all the dogs suffering from HQW compared to the control may be considered as a defense mechanism of the cortical neurons against the increase in the production of superoxide anions during the current state of oxidative stress. Increase in SOD activity might be attributed to up-regulation in its synthesis to counteract free radicals. The increase in SOD level in the present study was in agreement with the findings of Sathya et al. (2007) in dystocia affected buffalo and Dimri et al. (2008a) in canine demodicosis.

Table 3. Effect of different treatments on CAT (U /mg Hb).

Group	Day 0	Day 3	Day 7	Day 14	Day 28
I	125.75^bB±1.38	124.75^dA±0.70	125.13^dB±0.99	125.37^dB±0.91	124.00^cA±0.92
II	91.37^aA±0.51	91.50^aB±0.53	94.00^aC±1.51	100.75^aD±0.70	113.00^aE±0.92
III	92.50b^aA±1.41	95.00^cB±2.13	101.75^bC±0.70	109.25^bD±0.88	116.12^cE±0.83
IV	92.00b^aA±1.85	93.25^bA±1.03	103.75^cB±1.28	114.38^cC±0.74	122.25^bD±1.03
^A–EValues in the same row without a common superscript letter are significantly different (P<0.05).
^a–dValues in the same column without a common superscript letter are significantly different (P<0.05).

On day 0, the erythrocytes' CAT level was significantly (P < 0.05) lower in all the dogs with HQW as compared to the healthy control. Thereafter, CAT exhibited a continuous significant (P < 0.05) increase at all intervals in all of the treated groups, with the exception of day 3 in group IV. The increase was greatest in group IV followed by groups III and II. The values differed significantly amongst all the groups at corresponding intervals except groups II and III on day 28. CAT is the main scavenger of H₂O₂ at high concentration. It catalyzes the conversion of H₂O₂ to H₂O and molecular oxygen (Dringen 2000). Increased activity of SOD might have resulted into increased H₂O₂ production and thereby increased utilization of CAT for converting H₂O₂ into H₂O. This may be the reason behind lower CAT activity in dogs suffering from HQW. An increase in SOD activity and a decrease in CAT activities were also reported in vitiligo patients (Hanzneci et al. 2005).

Table 4. Effect of different treatments on GSH (µmol/g Hb).

Group	Day 0	Day 3	Day 7	Day 14	Day 28
I	0.97^bD±0.008	0.96^cC ±0.009	0.95^dBC±0.007	0.95^dAB±0.007	0.94^dA±0.008
II	0.53^aA±0.010	0.54^aA±0.049	0.61^aB±0.051	0.70^aC±0.041	0.77^aD±0.008
III	0.53^aA±0.064	0.62^bB±0.059	0.68^bC±0.059	0.81^bD±0.007	0.87^bE±0.007
IV	0.53^aA±0.035	0.64^bB±0.008	0.80^cC±0.009	0.88^cD±0.011	0.92^cE±0.007
^A–EValues in the same row without a common superscript letter are significantly different (P < 0.05).
^a–dValues in the same column without a common superscript letter are significantly different (P<0.05).

In all HQW affected dogs, a significant (P<0.05) decrease in erythrocytes' GSH level was noticed on day 0 as compared with the healthy control. In all of the treated groups, GSH showed a continuous significant (P < 0.05) increasing trend at all intervals. The increase was greatest in group IV followed by groups III and II. The GSH level differed significantly amongst all of the groups at corresponding intervals, with the exception of day 3 where the values differed non-significantly between groups III and IV. GSH, one of the first line endogenous defense antioxidants, is a tripeptide with an active sulphydril (-SH) group and can react with different electrophilic compounds and effectively scavenge free radicals either directly or indirectly through enzymatic reactions, protecting the cells against oxidative damage (Fang et al. 2002). Lower levels of GSH in groups II, III and IV in dogs suffering from HQW might be due to its enhanced utilization to neutralize excess free radicals. Similar findings were also reported in canine demodicosis (Dimri et al. 2008a), canine sarcoptic mange (Camkerten et al. 2009) and caprine sarcoptic mange (De and Dey 2010).

After treatment began, a continuous decrease in SOD, increase in CAT and GSH activity in all the treated groups was due to the decreasing antioxidant requirement at subsequent intervals. This was due to a continuous decrease in oxidative stress following the treatment, as also evidenced by decreased LPO. Furthermore, the critical evaluation of the results indicated that, when used in combination with CDT, SWD was better at reducing oxidative stress than therapeutic ultrasound. The decrease in oxidative stress was in correlation with the rate of clinical recovery in the animals under study. All of the animals regained their normal postural reactions, except hopping reaction in hind limbs, by day 14 of the therapy. Hopping reaction was achieved in 12 dogs (two in group II, four in group III and six dogs in group IV) by day 14 and in the rest of them by day 28. That indicated that the recovery process was early in animals, subjected to SWD followed by therapeutic ultrasound, when used in conjunction with conventional treatment.

It was concluded that as an adjunct therapy, SWD had an edge over therapeutic ultrasound in reducing oxidative stress in dogs suffering from HQW.