Effects of glia metabolism inhibition on nociceptive behavioral testing in rats

Fluoroacetate has been widely used to inhibit glia metabolism in vivo. It has yet to be shown what the effects of chronic intrathecal infusion of fluoroacetate on nociceptive behavioral testing are. The effects of chronic infusion of fluoroacetate (5 nmoles/h) for 2 weeks were examined in normal rats. Chronic intrathecal fluoroacetate did not alter mechanical threshold (von Frey filaments), responses to supra-threshold mechanical stimuli (von Frey filaments), responses to hot (hot plate) or cool (acetone test) stimuli and did not affect motor performance of the animals, which was tested with rotarod. This suggests that fluoroacetate at appropriate dose did not suppress neuronal activity in the spinal cord.


a b s t r a c t
Fluoroacetate has been widely used to inhibit glia metabolism in vivo.
It has yet to be shown what the effects of chronic intrathecal infusion of fluoroacetate on nociceptive behavioral testing are. The effects of chronic infusion of fluoroacetate (5 nmoles/h) for 2 weeks were examined in normal rats. Chronic intrathecal fluoroacetate did not alter mechanical threshold (von Frey filaments), responses to suprathreshold mechanical stimuli (von Frey filaments), responses to hot (hot plate) or cool (acetone test) stimuli and did not affect motor performance of the animals, which was tested with rotarod.

Value of the data
The effects of chronic intrathecal infusion of fluoroacetate on nociceptive behavioral testing were tested in adult rats.
Intrathecal fluoroacetate administered for 2 weeks in rats did not alter responses to mechanical, hot or cool stimuli and did not affect motor performance assessed with rotarod.
Chronic intrathecal fluoroacetate at appropriate dose did not impair nociceptive behavioral responses in the normal rat.

Data
We investigated the effects of chronic intrathecal infusion of fluoroacetate on nociceptive behavioral testing in adult rats. Fluoroacetate did not change the profiles of responses to von Frey hair stimuli applied on hind paws (Fig. 1A), did not alter responses to the Dynamic Hot Plate test (Fig. 1B) and to the acetone test (Fig. 1C) and did not affect motor performance of the animals, which was tested with rotarod ( Fig. 1D).

Animals
Adult rats (150-175 g) were obtained from Janvier (France) and maintained in a controlled environment (lights on 07:00-19:00, 22°C) with food and water freely available. They were housed 2 per cage. All efforts were made to minimize the number of animals used. The experiments followed the ethical guidelines of the International Association for the Study of Pain and the European Community Council directive of 22 September 2010 (2010/63/EU). The project was approved by Bordeaux Ethical Committee (CEEA50) under No. 50120169-A. Rats were acclimatized for 4 days to the animal facility and for 5 days to manipulations and devices prior to behavioral studies.

Surgery and drugs
Fluoroacetate or vehicle (artificial cerebrospinal fluid, aCSF) was administered through intrathecal catheters that were surgically implanted above the lumbar part of the spinal cord in animals at day 0 under isoflurane anesthesia. Catheters were connected to an Alzet osmotic pump delivering 0.5 μL/h.

Behavioral studies
Mechanical allodynia and hyperalgesia were measured at the hind paw using score values calculated from responses to von Frey filament stimulation according to the method adapted from Ducourneau et al. [2] and described in [1]. The first mean score value above detection was 1.2 and was used to obtain the nociceptive response-threshold. Accordingly, this score value corresponds to the stimulus intensity at which more than 50% of withdrawal responses could be observed over 5 responses. The first von Frey hair value giving a mean score above 1.2 was considered the nociceptive response-threshold.
To quantify mechanical hyperalgesia, we used a hyperalgesia score calculated as the sum of the differences between values obtained for each von Frey hair after and before (day 0) surgery and taken from above the value of the initial nociceptive threshold [1].
To quantify mechanical allodynia, shifts in nociceptive response-threshold were measured. Heat allodynia and hyperalgesia were assessed by using a computer-controlled hot plate analgesia meter (Bioseb, France). Animals were placed on the dynamic hot plate at 30°C and the plate temperature increased up to 45°C with 1°C min À 1 increment. During each degree interval, we scored hind paw lickings and paw withdrawals. The sum of the paw lickings and paw withdrawals was used as index of nociceptive response [3]. For each recording day, nociceptive responses were expressed as percentage of the maximal nociceptive responses obtained at 45°C in control rats.
Cold allodynia was assessed by application of a drop of acetone via a syringe to the back of the plantar surface of the hind paw, alternating between ipsilateral and contralateral sides. A total of five applications were made to each hind paw with at least 5 min separating each. The number of withdrawals was expressed as a percentage of maximum response.
To investigate the motor effects of fluoroacetate, rats were tested on the accelerating rotarod. All values are expressed as mean 7SEM. To assess changes after drug administration unpaired or paired (as appropriate) t-tests were performed. Differences were considered significant at p o0.05. In the acetone test, responses of both hind paws were pooled together for statistical comparison.