We investigated acupuncture effects on 16 dogs with IVDD. Canine age ranged 3–16 years old. Breeds included Miniature Dachshund (8), Norfolk Terrier (2), Chihuahua (1), Miniature Schnauzer (1), French Bulldog (1), Maltese (1), Jack Russell Terrier (1), and Mixed (1). The dogs were divided into two groups by acupuncture effects (Table 1). Before treatments, authors gave written informed consents to all dog’s owners. Ethical approval for this study was obtained from Higashi Chiba Animal Medical Center (Togane City, Chiba) (R2-1). Authors confirm this study was conducted following best veterinary practice procedures.
Acupuncture treatment method applied in this study was based upon the traditional Chinese veterinary medicine and Japanese methods (Sawamura 2019). Acupuncture needles used in this study were as follows: 1) Seirin D-Type No.1. (φ0.16) x 15 mm, 2) D-Type No.3 (φ0.20) x 15 mm, 3) Kanaken Disposable Acupuncture φ0.16 x 30 mm, φ0.18 x 30 mm, and 4) Kanaken Disposable Acupuncture φ0.20 × 30 mm needles were applied.
Acupoints were GV14, GV20-01, BL18, BL23, BL26, BL28, BL40, ST36 (Sawamura 2019). Electric acupuncture was performed for 20 minutes using Lasper Ace manufactured by Kanaken Co., Ltd. Acupuncture needles are placed into acupuncture points and left for 15 minutes, whereas electric currents are passed along the needle as a form of low frequency pulse therapy. Acupuncture effects were evaluated as a grade improvement in the IVDD grading scale (Oji 2015). In classification of cervical spinal cord disorders, grade 1 has with neck pain without neurological abnormalities; grade 2 also has gait disturbances and neurological abnormalities; grade 3 also has difficulty standing and walking and neurological abnormalities. In classification of thoracolumbar spinal cord disorders, grade1 has no spinal cord dysfunction but back pain; grade 2 has paresis of hind limbs; grade 3 has severe paresis of hind limbs; grade 4 has hindlimb paralysis and deep pain sensation; grade 5 has paralysis of the hind limbs and no deep pain sensation (Oji 2015). Acupuncture effect has been evaluated for 2 months after treatment except sample No.14 and 15.
Blood sampling and assays of metabolites and enzymes
Blood (2.5 mL) was collected from jugular veins with heparin as anti-coagulant before and after acupuncture treatment, both within approximately 30 minutes. After centrifugation at 1,200rpm for 5 minutes at 4℃, plasma was obtained and stored at -40℃ until use for assay.
Measurements of Plasma metabolites concentrations and enzyme activities
Plasma pyruvate concentrations were measured by a previously described method (Czok and Lamprecht 1974) and lactate concentrations were measured with commercial kit (Lactate Assay Kit-WST, Dojindo, Tokyo, Japan). Plasma lactate dehydrogenase (LDH) (Kaloustian et al. 1969) and malate dehydrogenase (MDH) (Bergmeyer and Bernet 1974) activities were measured by previously described methods, respectively. The plasma MDH/LDH (M/L) ratio was calculated as MDH activities divided by LDH activities. Plasma LDH isozyme patterns were detected by the biphasic agarose gel electrophoresis method utilizing commercial Quickgel LD gels (Helena Laboratories, Saitama, Japan) (Hirakawa et al.2012). LDH fraction were assessed and analyzed using Quick Scan (Helena Laboratories, Saitama, Japan).
Results are presented as means ± standard deviation (SD). Statistical significance was determined by paired-T test. The significant level was set at P < 0.05.