Effects of Glycemic Regulation on Chronic Postischemia Pain

Abstract

Background

Ischemia-reperfusion (I/R) injuries consist of enhanced oxidative and inflammatory responses along with microvascular dysfunction following prolonged ischemia and reperfusion. Since I/R injuries induce chronic postischemia pain (CPIP) in laboratory animals, it is possible that surgical procedures utilizing prolonged ischemia may result in chronic postoperative pain. Glycemic modulation during ischemia and reperfusion could impact pain following I/R injury, as glucose triggers oxidative, inflammatory and thrombotic reactions, whereas insulin has anti-oxidative, anti-inflammatory and vasodilatory properties.

Methods

110 rats underwent a 3-h period of ischemia followed by reperfusion to produce CPIP. CPIP rats had previously been divided into 6 groups with differing glycemic-modulation paradigms: 1) normal feeding; 2) fasting; 3) fasting with normal saline administration; 4) fasting with dextrose administration; 5) normal feeding with insulin administration; and 6) normal feeding with dextrose and insulin administration. Blood glucose levels were assessed during ischemia and reperfusion in these separate groups of rats, and they were tested for mechanical and cold allodynia over the following 21 days (on days 2, 5, 7, 9, 12 and 21 post-I/R injury).

Results

I/R injury in rats with normoglycemia or relative hyperglycemia (groups 1, 4) led to significant mechanical and cold allodynia; conversely, relative hypoglycemia associated with insulin treatment or fasting (groups 2, 3, and 5) reduced allodynia induced by I/R injury. Importantly, insulin treatment did not reduce allodynia when administered to fed rats given dextrose (group 6).

Conclusion

Our results suggest that glycemic levels at the time of I/R injury significantly modulate postinjury pain thresholds in CPIP rats. Strict glycemic control during I/R injury significantly reduces CPIP pain and, and conversely, hyperglycemia significantly enhances it, which could have potential clinical applications especially in the surgical field.

Introduction

Numerous surgical procedures involve intentional ischemic states, in order to reduce blood loss and produce a “clean” surgical field, from orthopaedic procedures using a tourniquet to any surgery involving organ resection, repair or transplant requiring vascular clamps. Ischemia-reperfusion injury (I/R injury), a complex condition consisting of intracellular injury and injurious inflammatory phenomena^1–3, is a well-established postischemic consequence of a prolonged ischemic episode⁴. Chronic pain disorders and various painful syndromes can result from procedures involving ischemia^5,6, and several factors that can impact the extent of the I/R injury have been described, such as the duration of the ischemic period, the extent of the ischemic region and the type of tourniquet used^7,8.

Various animal models of I/R injury have been created to study the underlying pathophysiologic mechanisms of this phenomenon^9–12. Chronic postischemia pain (CPIP) is an animal model in which rats undergo hind paw ischemia for 3 h, induced by the application of a tight-fitting tourniquet around their ankle, followed by reperfusion⁹. This I/R injury has been demonstrated to result in chronic mechanical and cold allodynia lasting for at least 4 weeks after the insulting event⁹. In CPIP rats, well-described mechanisms of I/R injury have been demonstrated, such as increased oxidative and inflammatory responses^13,14 and microvascular dysfunction^13,15. Indeed, in CPIP rats, free-radical induced lipid peroxidation, increased levels of proinflammatory cytokines tumor necrosis factor-α, interleukin-6 and interleukin-1β, increased levels of proinflammatory transcription factor nuclear factor κB and microvascular dysfunction (arterial vasospasms, capillary slow flow/ no reflow) were demonstrated in the affected hind paw^13,15.

Factors that can decrease chronic post-ischemia pain following I/R injury are not well known. Glycemia modulation, at the moment of the insult (i.e., during I/R injury), could potentially impact the chronic pain outcome. Glucose induces profound oxidative and inflammatory changes at the cellular and molecular levels, by activating several pro-inflammatory transcription factors^16–19, and could potentially worsen CPIP. In contrast, insulin exerts vasodilatory and antiinflammatory effects, respectively by increasing the production of nitric oxide²⁰, and by suppressing proinflammatory cytokines synthesis^21–23, which could be potentially beneficial in alleviating CPIP symptomatology.

Glycemic modulation using insulin infusion has been demonstrated to be of benefit in reducing mortality and morbidity in surgical intensive care unit, in cardiac surgery and in patients with an acute myocardial infarction^24,25. Hyperglycemia has also been shown to worsen I/R injury in other organs, such as kidneys²⁶ and brain²⁷. Numerous studies have observed and described neuropathic pain as a consequence of chronic hyperglycemia, using diabetic animal models^28–30, but nothing is known about the role of acute glycemic modulation during an I/R injury, and its consequences on post-ischemia pain.

We hypothesise that hyperglycemia (relative hyperglycemia) at the time of the insult will be associated with an enhanced postischemia pain, whereas tight glycemic control (relative hypoglycemia) at the time of the injury will be associated with a reduced post-ischemia pain. The following study was aimed at testing this hypothesis in CPIP rats after modulating the glycemia of the animals during ischemia and reperfusion.

1. Materials and methods

1.1 Animals

The present study was conducted on male Long Evans rats (300–500 g, Charles River, Senneville, Quebec, Canada). A total of 110 rats were used for the study. Animals were housed in groups of 2–4, under controlled lighting (12 h light-dark cycle) and temperature conditions. Food and water were available ad libitum, except on the day before and on the day of the experimental procedures (see 1.3 Glycemic treatments section). The experiments performed in the present study were approved by the McGill Animal Care and Ethics committees, and were conformed to the ethical guidelines of the Canadian Council on Animal Care and the International Association for the Study of Pain³¹.

1.2 Hind paw ischemia and reperfusion

An I/R injury of the left hind paw was induced as previously described⁹. Rats were anesthetised for a period of 3 h with an intraperitoneal bolus (55 mg/kg) of sodium pentobarbital, followed by an intraperitoneal infusion of the anesthetic for 2 h (27.5 mg/kg/h). After induction of anesthesia, a Nitrile 70 Durometer O-ring (O-rings, Seattle, WA) with a 5.5 mm internal diameter was placed around the rat’s left hind paw, proximal to the ankle and left in place for 3 h. After a 3 h-period of ischemia, the O-ring was cut and normal blood flow to the injured limb was re-established. We have previously established using laser Doppler flowmetry that an O-ring of this diameter completely occludes blood flow to the hind paw¹³. A subset of animals (N = 50) underwent sham procedure. These rats received general anesthesia with sodium pentobarbital as in sentence 2, this paragraph, but were not fitted with an O-ring. Sixty minutes following the termination of pentobarbital infusion, the rats had recovered a normal respiratory pattern, and could be safely returned to their housing cages.

1.3 Glycemic treatments

In order to study the effect of glycemic modulation on chronic postischemia pain, 6 groups of rats were submitted to different glycemic conditions. Glycemia was manipulated - raised, lowered or unaltered - in order to make comparisons between groups of rats with differing mean glycemia. Fifteen to 21 rats were included in each group. In the regular diet (fed) group, animals had unlimited access to food and water prior to anesthesia. The second group of animals (fasted) had its access to food withdrawn 18 h prior to anesthesia, but had unlimited access to water. The third group of animals (fasted + ns) had their access to food withdrawn 18 h prior to anesthesia, but had unlimited access to water; these rats received three intraperitoneal injections of 1 mL of normal saline (ns): at baseline, and at 60 and 120 min following the start of ischemia. The fourth group of rats (fasted + dextrose) had their access to food withdrawn 18 h prior to anesthesia, but had normal access to water; these rats received three intraperitoneal injections, each of 1 mL, of dextrose dissolved in deionised water (final concentration: 40%, w/v, from now on referred to as DW40%): at baseline, and at 60 and 120 min following the start of ischemia. The fifth group of rats (fed + insulin) were normally fed, but were administered subcutaneous injections of insulin R diluted in normal saline, according to a sliding scale we created for this experiment (glycemia at baseline ≤ 6: 0.5 international units (IU)/kg; > 6 ≤ 7: 1 IU/kg; > 7 ≤ 8: 1.5 IU/kg; > 8 ≤ 9: 2 IU/kg; > 9 ≤ 10: 2.5 IU/kg; because none of the rats had a baseline glycemia > 10, it was not necessary to further extend the insulin sliding scale). The total dose of insulin was administered in two half-doses, the first one immediately following the onset of ischemia, and the second 60 min after induction of ischemia. The maximal volume of insulin administered was 50 μL. The last group of rats (fed + insulin + dextrose) were normally fed and received insulin according to a sliding scale (as group 5), at the same time points. However, they were also administered intraperitoneal DW40% (as group 4), in order to maintain their glycemia above the baseline value.

1.4 Glucose measurements

Glycemic levels were measured with a glucometer FreeStyle mini (Abbott Diabetes Care, Mississauga, Ontario, Canada). Blood was withdrawn from the extremity of the rat tail, from a submillimetric incision, by an observer blinded to treatment. Volume of blood withdrawn varied between 0.3 and 0.5 ml. The glycemia of the rats was regularly monitored during the ischemic period and the first hour of reperfusion. Blood samples were withdrawn at baseline, and then 30, 90, and 150 min after induction of ischemia, as well as 20, 40, and 60 min following reperfusion.

1.5 Mechanical and thermal sensitivity

1.5.1 Hind paw mechano-allodynia

Mechanical allodynia was assessed by measuring the 50% withdrawal response to von Frey filaments, according to a modification of a previously published method³², by an observer blinded to treatment. The animals were placed in Plexiglas® cages with a wire grid bottom. Von Frey filaments (Stoelting, Wood Dale, IL) were applied to the plantar surface of the hind paw for 10 s or less if the rat responded to the stimulus, by withdrawing, flicking, stamping or licking its paw. Filaments were applied in ascending or descending order of strength to determine the filament closest to the response threshold. The minimal stimulus intensity was 0.25 g, whereas the maximal intensity was 15 g.

1.5.2 Hind paw cold allodynia

Cold allodynia was evaluated by exposing the rats’ paw to a drop of acetone, using a modification of a previously published method³³, by an observer blinded to treatment. A drop of acetone was applied on the plantar surface of the heel, and the rat’s response was observed for the following 20 s. Normal rats either ignore the stimulus or respond with a rapid, brief withdrawal. The intensity and duration of the rats’ responses to the nociceptive stimulus were scored according to the following rating scale⁹: 1 = rapid paw flicking, stamping, or shaking, less than 1 s; 2 = repeated paw stamping, shaking, or paw lift less than 3 s; 3 = above behaviors or hind paw licking for more than 3 s. Acetone was applied 3 times over 3 min, and the 3 trials were then averaged.

1.6 Statistical analysis

Glucose levels (mmol/L), paw withdrawal thresholds (g) and acetone scores (nociceptive cold scores) were expressed as mean ± 95% confidence limits. Statistical differences between glycemic values at baseline before ischemia and at six subsequent points in time (i30, i90, i150, r20, r40, and r60; i = ischemia, r = reperfusion) were assessed with Tukey’s post hoc tests following a three-way analysis of variance (ANOVA) (glycemic condition by injury (CPIP or sham CPIP) by time as the repeated factor). Statistical differences between groups, collapsed over time on mechanical and cold allodynia of the ipsilateral and contralateral hind paws were similarly assessed using a mixed model repeated measure ANOVA, and Tukey’s post-hoc tests. Comparisons were considered significant if P < 0.05 (two-tailed tests in all cases). Correlations between glucose levels (averaged over the ischemia, reperfusion or ischemia and reperfusion periods) and von Frey or acetone cold scores (averaged for each subject over the entire behavioural testing period) were calculated using the Spearman rank correlation coefficient (ρ). The coefficients of determinations were obtained using the r of Pearson. Differences between correlation coefficients were assessed using the Z score. Statistics were computed with Statistica (Version 6, Statsoft, Tulsa, OK), and graphs were prepared with GraphPad Prism 5.00 (GraphPad Software, Inc., 2007, La Jolla, CA).

2. Results

2.1 Glycemic measurements

Three way repeated measures ANOVA (see table 1) revealed a non-significant main effects of injury (i.e., sham or CPIP) on glucose level, and main effect analysis indicated that there were no significant differences in the mean glucose levels between the CPIP groups and their sham control groups for the fed animals (6.75 ± 0.40 vs. 6.83 ± 0.57), the fasted + ns animals (4.62 ± 0.46 vs. 4.65 ± 0.57), the fed + insulin animals (5.62 ± 0.42 vs. 5.50 ± 0.44), the fasted + dextrose animals (7.25 ± 0.46 vs. 6.96 ± 0.44), the fed + dextrose + insulin animals (7.66 ± 0.49 vs. 7.65 ± 0.46) and the fasted group (4.75 ± 0.42 vs. 5.42 ± 0.44) (all P > 0.05, Tukey’s tests).

Table 1.

Analysis of Variance Table for Glucose Measurements.

Effect	SS	df	MS	F	p
Glycemic condition	788.52	5	157.70	54.737	0.000000
Injury	0.28	1	0.28	0.096	0.757888
Condition X Injury	21.34	5	4.27	1.482	0.203222
Error	270.83	94	2.88
Time	22.49	6	3.75	4.303	0.000296
Time X Condition	558.03	30	18.60	21.352	0.000000
Time X Injury	9.52	6	1.59	1.821	0.092723
Time X Condition X Injury	106.00	30	3.53	4.056	0.000000
Error	491.34	564	0.87

Glycemic condition and injury are independent groups factors and time is a repeated factor

df = degrees of freedom; F = F statistic; MS = mean square; p = p value; SS = Sum of squares.

Time course analysis with post hoc pair-wise comparisons, depicted in figure 1 showed that glucose levels were significantly increased over baseline in the fasted + dextrose and fed + dextrose + insulin groups, and significant decreased in the fasted, fasted + ns, and fed + ins groups, at the times indicated by the asterisks in figure 1 (* P < 0.05; ** P < 0.01, Tukey’s test). There were also significant differences in glucose levels between the fed and the fasted groups (†P < 0.01, post-hoc Tukey’s), the fasted + ns and the fasted + dextrose groups (§ P < 0.01), the fed and the fed + insulin groups (‡ P < 0.01), and between the fed + insulin and the fed + dextrose + insulin groups (# P < 0.01) at the times indicated by the above symbols (post hoc Tukey’s). Generally glucose levels varied between glycemic conditions to a greater degree during ischemia than during reperfusion, as documented by the significant glycemic condition by time interaction (see table 1).

Fig. 1.

Mean (mean ± 95% confidence limits) glucose levels at different time points during the ischemic (i) and reperfusion (r) periods for rats with chronic postischemic pain in the fed (N = 12), fasted (N = 11), fasted + normal saline (NS; N = 11), fasted + dextrose (dex; N = 8), fed + insulin (ins; N = 9) and fed + dex + ins (N = 9) groups. The glucose levels of the fasted animals were lower than the glucose levels of the fed animals throughout the ischemia and reperfusion periods († P < 0.01), as well as lower than their baseline at times indicated by asterisks (* P < 0.05; ** P < 0.01). Dextrose administration to the fasted + dex group increased glucose levels compared to their baseline levels during ischemia (** P < 0.01) and compared to the fasted + NS group throughout the ischemia and reperfusion periods (§ P < 0.01). The fed + dex + ins had significantly higher glucose levels than the fed + ins groups at the times indicated by the symbols (# P < 0.01). Insulin received by the fed + ins group lowered the glucose levels compared to their baseline at times indicated by asterisks (* P < 0.05; ** P < 0.01), or to the fed group during the ischemic period (‡ P < 0.01).

2.2 Mechanical and thermal hypersensitivity

2.2.1 Mechanical thresholds

2.2.1.1 Ipsilateral paw withdrawal thresholds

Generally, CPIP animals developed significantly lower ipsilateral paw withdrawal thresholds (PWTs) than shams. Three way repeated measures ANOVA (see table 2) revealed a significant main effect of injury (i.e., sham or CPIP). Time course analysis with post hoc pairwise comparisons, depicted in figure 2, showed that the ipsilateral PWTs of CPIP rats were lower than sham rats on each testing day for rats in the fed and fasted + dex glycemic conditions, and for four of the six test days for the fed + dex + ins glycemic condition (** P < 0.01, Tukey’s test).

Table 2.

Analysis of Variance Table for Ipsilateral Paw-Withdrawal Thresholds.

Effect	SS	df	MS	F	p
Glycemic condition	1378.42	5	275.68	13.659	0.000000
Injury	4296.72	1	4296.72	212.882	0.000000
Condition X Injury	548.22	5	109.64	5.432	0.000180
Error	2058.72	102	20.18
Time	158.75	5	31.75	3.645	0.003000
Time X Condition	231.86	25	9.27	1.065	0.380037
Time X Injury	121.44	5	24.29	2.788	0.016978
Time X Condition X Injury	478.93	25	19.16	2.199	0.000801
Error	4442.82	510	8.71

Glycemic condition and injury are independent groups factors and time is a repeated factor.

df = degrees of freedom; F = F statistic; MS = mean square; p = p value; SS = Sum of squares.

Fig. 2.

Time-course of ipsilateral paw withdrawal thresholds (PWTs) (mean ± 95% confidence limits) for the fed, fasted, fasted + normal saline (NS), fasted + dextrose (dex), fed + insulin (ins) and fed + dex + ins groups. Ipsilateral PWTs of rats with chronic postischemia pain were lower than sham rats on each testing day for rats in the fed and fasted + dex glycemic conditions, and on test days 2–7 and 21 for the fed + dex + ins glycemic condition (** P < 0.01, Tukey’s test)

In order to examine the highly significant glycemic condition X injury interaction (see table 2) we plotted PWTs grouped by glycemic condition and injury averaged over time for the ipsilateral hind paw (fig. 3A) and contralateral hind paw (fig. 3B). Ipsilateral PWTs were significantly lower for CPIP rats compared to shams in five of the six glycemic conditions (i.e., fed, fasted, fasted + dex, fed + ins, fed + dex + ins, but not in the fasted + ns condition (fig. 3A) (* P < 0.05; ** P < 0.01; Tukey’s tests). When comparing CPIP rats across condition, the fed rats exhibited significantly lower PWTs compared to the fasted group (§ P < 0.01), and. the fasted + dextrose group of rats exhibited significantly lower PWTs than fasted + ns rats (# P < 0.01). The fed CPIP rats also showed significantly lower PWTs than did the fed + insulin rats (‡ P < 0.01). The PWTs of the fed + dextrose + insulin group of CPIP rats were significantly lower than those of the fed + insulin group (‡ P < 0.01) (all Tukey’s tests).

Fig. 3.

Mean (mean ± 95% confidence limits) ispilateral (A) and contralateral (B) paw withdrawal thresholds (PWTs, g) averaged across test days for sham rats (N’s follow each group) and rats with chronic post-ischemia pain (CPIP) (N’s indicated in fig. 1) in the fed (N = 6), fasted (N = 10), fasted + normal saline (NS; N = 10), fasted + dextrose (dex; N = 8), fed + insulin (ins; N = 10) and fed + dex + ins (N = 6) groups. A. For each glycemic treatment, PWTs of CPIP rats were lower than their corresponding sham rats (* P < 0.05, ** P < 0.01). PWTs of CPIP fed rats were significantly lower than those of CPIP fasted rats (§ P < 0.01) or fed + ins rats (‡ P < 0.01). PWTs of CPIP fed + dex + ins rats were significantly lower than those of CPIP fed + ins rats (‡ P < 0.01). PWTs of CPIP fasted + dex rats were also significantly lower than those of CPIP fasted + NS rats (# P < 0.01). B. PWTs of the CPIP rats were significantly lower than shams for only the fed, fasted + dex and fed + dex + ins groups (** P < 0.01). PWTs of the CPIP fed + dex + ins rats were significantly lower than those of CPIP fed + ins rats († P < 0.01) and fed rats (§ P < 0.01). PWTs of the CPIP fasted + dex rats were significantly lower than CPIP fasted + NS rats (#P < 0.01).

2.2.1.2 Contralateral PWTs

CPIP rats generally appeared to exhibit lower contralateral PWTs than shams, with greater effects for the fasted + dex and fed + dex + ins conditions. Three way repeated measures ANOVA (see table 3) revealed a significant main effect of injury (i.e., sham or CPIP). However, time course analysis with post hoc pair-wise comparisons, depicted in figure 4, showed that the contralateral PWTs of CPIP rats were not significantly lower than sham rats on any testing day for rats in all glycemic conditions (Tukey’s test).

Table 3.

Analysis of Variance Table for Contralateral Paw-Withdrawal Thresholds.

Effect	SS	df	MS	F	p
Glycemic condition	585.16	5	117.03	4.734	0.000626
Injury	1417.38	1	1417.38	57.334	0.000000
Condition X Injury	446.40	5	89.28	3.611	0.004763
Error	2521.59	102	24.72
Time	155.27	5	31.05	3.314	0.005904
Time X Condition	211.74	25	8.47	0.904	0.600716
Time X Injury	49.52	5	9.90	1.057	0.383477
Time X Condition X Injury	243.28	25	9.73	1.038	0.413632
Error	4779.32	510	9.37

Glycemic condition and injury are independent groups factors and time is a repeated factor.

cf = degrees of freedom; F = F statistic; MS = mean square; p = p value; SS = Sum of squares.

Fig. 4.

Time-course of contralateral paw withdrawal thresholds (PWTs) (mean ± 95% confidence limits) for the fed, fasted, fasted + normal saline (NS), fasted + dextrose (dex), fed + insulin (ins) and fed + dex + ins groups. Contralateral PWTs of rats with chronic postischemia pain were not significantly lower than sham rats on any testing day for rats in all glycemic conditions.

Once again to examine the highly significant glycemic condition X injury interaction (see table 3), we plotted PWTs grouped by glycemic condition and injury averaged over time for the contralateral hind paw (fig. 3B). PWTs were significantly lower for CPIP rats compared to shams in three of the six glycemic conditions (i.e., fasted + dex, fed + ins, fed + dex + ins, but not in the fed, fasted or fasted + ns conditions (* P < 0.05; ** P < 0.01; Tukey’s tests). Although the differences were not as extensive as for ipsilateral PWTs, contralateral PWTs of CPIP rats also differed significantly between glycemic conditions. Thus, the fed + dextrose + insulin group had significantly lower PWTs than the fed + ins group († P < 0.01) and the fed group (§ P < 0.01). The fasted + dextrose group also exhibited significantly lower contralateral PWTs than the fasted + ns group (# P < 0.01) (all Tukey’s test).

As displayed in the scatterplot of ipsilateral PWTs versus glucose levels in figure 5A, relatively hyperglycemic CPIP animals have lower PWTs than relatively hypoglycemic CPIP animals. Indeed, there is a negative correlation (rs = −0.679, P = 0.000019) between the mean PWTs and glucose levels measured during the entire I/R injury period across all groups. Thus, 46.1% of the observed variation in the ipsilateral PWTs depends on glycemia. This negative correlation is also significant when using glucose levels from only the ischemic period (rs = −0.704, P = 0.000022) or from only the reperfusion period (rs = −0.588, P = 0.015), with 49.4% (fig. 5B) and 34.6% (fig. 5C) of the observed variation in ipsilateral PWTs depending on the glycemic levels measured during these periods, respectively. The correlation between PWTs and glucose levels during ischemia is not statistically different than the correlation between PWTs and glucose levels during reperfusion (Z = 0.783, P > 0.05).

Fig. 5.

Scatterplot illustrating the relationship between ipsilateral paw withdrawal thresholds and glycemia in rats with chronic postischemia pain (CPIP) using glucose levels for the entire I/R injury (A), for only the ischemic period (B) or only the reperfusion period (C). Specific groups in the scatterplot are identified in the symbol legend. For each plot there was a significant negative correlation between the paw withdrawal thresholds and the mean glucose level (mmol/L) during I/R injury for CPIP rats (R² = coefficient of determination).

2.2.2 Cold scores

2.2.2.1 Ipsilateral cold scores

Generally, CPIP animals developed significantly higher ipsilateral cold scores than shams. Three way repeated measures ANOVA (see table 4) revealed a significant main effect of injury (i.e., sham or CPIP). Time course analysis with post hoc pair-wise comparisons, depicted in figure 6, showed that the ipsilateral cold scores of CPIP rats were higher than sham rats on 3 of the testing day for rats in the fed glycemic condition, and for 2 of the test days for the fasted + dex glycemic condition (Tukey’s test).

Table 4.

Analysis of Variance Table for Ipsilateral Nociceptive Cold Scores.

Effect	SS	df	MS	F	p
Glycemic condition	39.3596	5	7.8719	9.408	0.000000
Injury	87.5721	1	87.5721	104.656	0.000000
Condition X Injury	13.8772	5	2.7754	3.317	0.008494
Error	76.1453	91	0.8368
Time	22.1783	5	4.4357	14.770	0.000000
Time X Condition	9.8359	25	0.3934	1.310	0.146277
Time X Injury	1.2454	5	0.2491	0.829	0.529180
Time X Condition X Injury	13.4952	25	0.5398	1.797	0.011062
Error	136.6437	455	0.3003

Glycemic condition and injury are independent groups factors and time is a repeated factor.

df = degrees of freedom; F = F statistic; MS = mean square; p = p value; SS = Sum of squares.

Fig. 6.

Time-course of ipsilateral nociceptive cold scores (mean ± 95% confidence limits) for the fed, fasted, fasted + normal saline (NS), fasted + dextrose (dex), fed + insulin (ins) and fed + dex + ins groups. Ipsilateral cold scores of rats with chronic postischemia pain were higher than sham rats on test days 5, 9, and 12 for rats in the fed glycemic condition, and on test days 7 and 12 for the fasted + dex glycemic condition (** P < 0.01, Tukey’s test).

In order to examine the highly significant glycemic condition X injury interaction (see table 2) we plotted cold scores grouped by glycemic condition and injury averaged over time for the ipsilateral hind paw (fig. 7A) and the contralateral hind paw (fig. 7B). Ipsilateral nociceptive cold scores were significantly higher for CPIP rats compared to shams in three of the six glycemic conditions (i.e., fed, fasted + dex, fed + dex + ins, but not in the fasted, fasted + ns or fed + ins conditions (fig. 7A) (* P < 0.05; ** P < 0.01; Tukey’s tests). When comparing CPIP rats across condition, the CPIP fed group exhibited significantly increased cold scores compared to the CPIP fasted group (‡ P < 0.01). When compared to the fed + insulin group, the fed († P < 0.01) and the fed + dex + ins († P < 0.01) groups exhibited significantly higher cold scores. However, there was no significant difference between the fasted + ns group and the fasted + dextrose group (P > 0.05).

Fig. 7.

Mean ± 95% confidence limits) ipsilateral (A) and contralateral (B) nociceptive cold scores averaged across test days for sham rats and rats with chronic post-ischemia pain (CPIP) in the fed, fasted, fasted + normal saline (NS), fasted + dextrose (dex), fed + insulin (ins) and fed + dex + ins groups. A. For the fed, fasted + dex and fed + dex + ins glycemic conditions, the ipsilateral nociceptive cold scores were significantly greater in CPIP rats than shams (** P < 0.01). Nociceptive cold scores of CPIP fed rats were significantly higher than those of fasted rats (‡ P < 0.01) and fed + ins rats († P < 0.01), and the cold scores of CPIP fed + dex + ins rats were significantly higher than those of fed + ins rats († P < 0.01). B. The contralateral nociceptive cold scores were significantly greater in CPIP rats than shams only for rats in the fed + dex + ins groups (** P < 0.01). Nociceptive cold scores of CPIP fed + dex + ins rats were also significantly higher than those of CPIP fed + ins rats († P < 0.01) and CPIP fed rats (§ P < 0.01).

2.2.2.2 Contralateral cold scores

CPIP rats generally appeared to exhibit higher contralateral cold scores than shams, with greater effects for the fasted + ns and fed + dex + ins conditions. Three way repeated measures ANOVA (see table 5) revealed a significant main effect of injury (i.e., sham or CPIP). Time course analysis with post hoc pair-wise comparisons, depicted in figure 8, showed that the contralateral PWTs of CPIP rats were significantly higher than sham rats on only one test day for rats in the fasted + ns and fed + dex + ins glycemic conditions (* P < 0.05, ** P < 0.01, Tukey’s test).

Table 5.

Analysis of Variance Table for Contralateral Nociceptive Cold Scores.

Effect	SS	df	MS	F	p
Glycemic condition	16.2914	5	3.2583	7.7532	0.000004
Injury	15.1510	1	15.1510	36.0524	0.000000
Condition X Injury	4.1897	5	0.8379	1.9939	0.086940
Error	38.2426	91	0.4202
Time	18.1947	5	3.6389	20.1541	0.000000
Time X Condition	7.6965	25	0.3079	1.7051	0.019053
Time X Injury	4.1819	5	0.8364	4.6323	0.000391
Time X Condition X Injury	8.2141	25	0.3286	1.8197	0.009673
Error	82.1529	455	0.1806

Glycemic condition and injury are independent groups factors and time is a repeated factor.

df = degrees of freedom; F = F statistic; MS = mean square; p = p value; SS = Sum of squares.

Fig. 8.

Time-course of contralateral nociceptive cold scores (mean ± 95% confidence limits) for the fed, fasted, fasted + normal saline (NS), fasted + dextrose (dex), fed + insulin (ins) and fed + dex + ins groups. Contralateral PWTs of rats with chronic postischemia pain were significantly higher than sham rats only on test day 5 for rats in the fasted + ns condition and on test day 21 in the fed + dex + ins glycemic condition (* P < 0.05, ** P < 0.01, Tukey’s test).

Once again we plotted nociceptive cold scores grouped by glycemic condition and injury averaged over time for the contralateral hind paw (fig. 7B). Nociceptive cold scores were significantly higher for CPIP rats compared to shams in only the fed + dex + ins glycemic condition (fig. 3A) (** P < 0.01; Tukey’s tests). Furthermore for CPIP rats, the fed + dextrose + insulin group exhibited significantly higher contralateral cold scores than the fed + insulin group († P < 0.01) and the fed group (§ P < 0.01). There was no significant difference between the contralateral cold scores of the fasted + dextrose group compared to the fasted + ns group (P > 0.05).

As displayed in the plot of ipsilateral cold scores versus glucose levels in figure 9A, relatively hyperglycemic CPIP animals have higher cold scores than relatively hypoglycemic CPIP animals. Thus, there is a positive correlation (rs = 0.490, P = 0.0055) between the mean cold scores and the glucose levels measured during the entire I/R. Thus, 24% of the observed variation in the acetone scores depends on glycemia. This positive correlation is also significant when using glucose levels from only the ischemic period (rs = 0.528, P = 0.0203) or only the reperfusion period (rs = 0.419, P < 0.0222), with 27.9% (fig. 9B) and 17.5% (fig. 9C) of the observed variation in ipsilateral cold responses depending on the glucose levels measured during these periods, respectively. The correlation between ipsilateral cold scores and glucose levels during ischemia is not statistically different than the correlation between ipsilateral cold responses and glucose levels during reperfusion (Z = 0.456, P > 0.05). Consequently, the glycemia present during these two periods similarly influence the ipsilateral cold allodynia exhibited after I/R injury.

Fig. 9.

Scatterplots illustrating the relationship between ipsilateral nociceptive score and glycemia in rats with chronic postischemia pain (CPIP) using glucose levels for the entire I/R injury (A), for only the ischemic period (B) or only the reperfusion period (C). Specific groups in the scatterplot are identified in the symbol legend. For each period (ischemia and reperfusion), there was a significant positive correlation between the nociceptive cold score and the mean glucose value (mmol/L) during I/R injury for CPIP rats (R² = coefficient of determination).

3. Discussion

This study has demonstrated that manipulating glycemia during I/R injury significantly influences the degree of allodynia in CPIP rats. Thus, CPIP animals with a higher glucose, such as the normally fed animals and the animals receiving DW40%, exhibited significantly higher mechanical and cold allodynia than the animals with lower glycemic levels, such as the fasted rats and fed rats which were administered insulin. These results strongly suggest a protective role for relative hypoglycemia during I/R injury.

3.1 The effect of glucose on post-ischemia pain

Hyperglycemia can increase oxidative stress by several mechanisms. Thus, in a hyperglycemic state, the production of reactive oxygen species such as the superoxide radical is increased^27,34,35. Additionally, the levels of endogenous antioxidants, such as α-tocopherol, are decreased by hyperglycemia^36–38. Along with oxidative stress, hyperglycemia activates several pro-inflammatory transcription factors, such as nuclear factor κB¹⁶, activator protein 1¹⁷, and early growth response protein 1¹⁷. The activation of these transcription factors leads to increased production of several proinflammatory mediators, such as cytokines, chemokines, cell adhesion molecules, inflammatory enzymes and some acute phase proteins^39–42, which amplify the inflammatory process.

Hyperglycemia has previously been shown to increase the levels of proinflammatory mediators in a rat model of cerebral I/R, exacerbating the ischemic insult³⁹. Thus, it is possible that hyperglycemic rats in our study suffered from an increased ischemic insult, the burden of which was added to the oxidative and inflammatory states. Although the relative contribution of these three pathological phenomena is yet to be determined, their increases are likely to explain the enhanced cold and mechanical allodynia we observed.

Another potential contributory mechanism which could have led to altered pain threshold in hyperglycemic rats could be related to thrombotic phenomena. Hence, glucose plays a role in the activation of a cascade ultimately leading the production of tissue factor, which creates a prothrombotic state^43–46. Thus, increasing thrombotic susceptibility in a context of increased oxidative and inflammatory stresses can lead to a vicious circle exacerbating ischemic injury.

An additional mechanism by which hyperglycemia can lead to an alteration in pain threshold could lie in an up-regulation of the kinin B₁ receptor, the levels of which were demonstrated to increase in a rat model of insulin resistance⁴⁷. Kinins are proinflammatory mediators of pain associated with nociception^48, and the B₁ receptor is involved in inflammatory responses leading to hyperalgesia^49,50.

3.2 The effect of insulin on postischemia pain

Fed animals which were administered insulin have less allodynia than the fed animals that did not receive insulin. Additionally, fed animals that received both DW40% and insulin exhibited more allodynia than the fed animals that received insulin without DW40%. The difference in pain thresholds for the latter two groups, could be due to two factors. By lowering glycemia, insulin could counteract the oxidative, inflammatory, and thrombotic cascades which occur during a hyperglycemic state, or alternatively insulin could itself exert intrinsic antiinflammatory, antioxidative, and antithrombotic effects^{17,22,51–56}. However, considering that insulin was administered to these two groups according to the same sliding scale, and that the only variable changed was the supplemental dextrose (which clearly increased blood glucose), the results support the hypothesis that insulin administration per se is not as protective as lowering glycemic levels.

3.4 Contralateral effects

Significant contralateral allodynia has been described for the normally fed CPIP animals⁹ and in various models of neuropathic and inflammatory pain⁵⁷. The accepted explanation for these contralateral effects is central sensitisation caused by the peripheral activation of C-afferent nociceptive fibres which modify the functional response of the spinal cord neurons to other inputs applied after the conditioning input⁵⁸. Interestingly, in our experiments, the fed + dextrose + insulin CPIP rats exhibited significantly lower PWTs and higher cold scores on the contralateral side than fed CPIP rats, which suggests that central sensitization was more prominent in these rats. To explain this observation, we hypothesize that hyperglycemia associated with the administration of dextrose leads to more a significant ischemic injury and increased central inputs, which in turn increase central sensitisation. A second possibility (although unsupported by previous literature) is that dextrose itself has central effects which enhance pain facilitation.

3.5 Potential clinical applications

The observation that glycemic modulation affects postischemia pain has clinical implications, especially in the operating room. Post-ischemia pain and painful syndromes such as complex regional pain syndrome type-I can theoretically follow any surgical procedure or trauma involving prolonged ischemia, such as open reduction and internal fixation of fractures, arthroscopic surgery, etc. The occurrence and chronicity of postischemia pain could potentially be significantly reduced by intra-operative maintenance of strict glycemic control, potentially by an insulin infusion as patients are normally already fasting before a surgery, keeping in mind, of course, the deleterious effects of severe hypoglycemia. It is also tempting to extrapolate that dextrose infusions during a surgical procedure may contribute to postoperative pain, and should therefore be avoided if possible.

4. Concluding remarks

Our investigations point to a critical role of glycemia in CPIP, with significantly increased pain sensitivity when animals have higher glycemic levels at the time of the injury, and significantly lower pain sensitivity when animals have lower glycemic levels at the time of the injury, whether this is achieved by fasting or by insulin administration. As mentioned in the discussion, we believe that the prooxidative, inflammatory, and thrombotic properties of hyperglycemia play a critical role in leading to enhanced allodynic/hyperalgesic states. Of interest, our results demonstrate that higher glycemic levels at the time of I/R injury also lead to enhanced pain sensitivity in the contralateral limb, suggesting that hyperglycemia might play a role in the development of central sensitisation. Our results might lead to several interesting clinical applications, most notably in the surgical field. Hence, it is tempting to extrapolate that maintaining strict glycemic levels during a surgical procedure might lead to a reduction in postsurgical pain.