Nitroxidergic modulation of behavioural, cardiovascular and immune responses, and brain NADPH diaphorase activity upon morphine tolerance/dependence in rats

Figures & data

Table 1. Score-point scale of opioid dependence/withdrawal symptoms.

Symptoms	Score points	Maximum score points/10 min
Defecation (normally formed stool)	1 for each 4 droppings	2
Salivation, lacrimation	1 for each	2
Back-fur piloerection, ptosis	1 for each leastwise 60 s non-stop	5
Sniffing, grooming, gnawing	1 for each leastwise 60 s non-stop	5
Teeth chattering/mastication/chew	1 for each leastwise 60 s non-stop	5
Penile licking	1 for leastwise 30 s non-stop	5
Vocalization on touch at 2 min	1 for each episode	5
Aggressive behaviour on touch at 2 min	1 for each episode	5
Post-treatment body weight loss	1 for each 2.5% bw loss on next day	8
Escape attempts	1 for each 2 attempts	8
Wet-dog shakes	1 for each leastwise 60 s non-stop	8
Hyperactivity, exploration, rearing	1 for each leastwise 60 s non-stop	8
Writhing/wrench/twist	1 for each	8
Diarrhoea (loosely formed stool)	1 for each episode	8
Wax-figure posture	1 for each leastwise 60 s non-stop	10
Prostration	1 for each leastwise 30 s non-stop episode	10

Table 2. Quantitative evaluation of behavioural symptoms upon various drug treatments.

Treatment (group)	Points/10 min	Points/30 min
Chronic morphine (G1)	17.5 ± 1.1^a	42.5 ± 6.8^a
Chronic L-NAME (G2)	6.0 ± 0.9	17.0 ± 3.7
Chronic morphine plus L-NAME (G3)	6.0 ± 0.9	17.0 ± 4.2
Morphine withdrawal (G5)	47.0 ± 4.3^b	126.0 ± 19.3^b
L-NAME plus morphine withdrawal (G6)	14.5V 2.4^c,d	39.5 ± 7.4^c,d
Acute morphine (G7)	7.2 ± 1.2	19.0 ± 6.1
Acute naloxone (G8)	4.5 ± 0.4	13.5 ± 1.9

^aP ≤ 0.01 vs. G4; ^bP ≤ 0.001 vs. G1; ^cP ≤ 0.05 vs. G4; ^dP ≤ 0.001 vs. G5.

Table 3. Changes (%) in systolic blood pressure (SBP), diastolic blood pressure (DBP), mean blood pressure (MBP) and heart rate (HR) due to first (I) and second (II) morphine application in the absence (D1) and presence (D2) of L-NAME.

	I (D1)	II (D1)	I (D2)	I (D2)
SBP	−39 ± 9	2 ± 1^a	−46 ± 7	−39 ± 10
DBP	−59 ± 10	1 ± 2^a	−58 ± 10	−52 ± 12
MBP	−52 ± 10	1 ± 2^a	−52 ± 6	−46 ± 12
HR	−78 ± 6	−13 ± 6^a	−89 ± 1	−75 ± 8

^aP ≤ 0.001 vs. I (G1).

Table 4. Changes (%) in systolic blood pressure (SBP), diastolic blood pressure (DBP), mean blood pressure (MBP) and heart rate (HR) due to first (I) and second (II) morphine application in the absence (D3) and presence (D4) of L-NAME and pretreatment with L-arginine.

	I (D3)	II (D3)	I (D4)	I (D4)
SBP	−42 ± 9	7 ± 2^a	−42 ± 4	−21 ± 6^b
DBP	−58 ± 8	6 ± 4^a	−53 ± 7	−29 ± 9^b
MBP	−53 ± 7	7 ± 3^a	−50 ± 10	−26 ± 6^b
HR	−81 ± 6	−26 ± 10^a	−71 ± 9	−62 ± 10^b

^aP ≤ 0.001 vs. I (G3); ^bP ≤ 0.05 vs. I (G4).

Table 5. Effect of chronic morphine and L-NAME treatment on lymphocyte proliferation.

Concanavalin A	Group G1	Group G2	Group G3	Group G4
125 μg/ml	2.74 ± 0.29^a	117.62 ± 14.63^a	28.84 ± 3.58	38.75 ± 3.77
500 μg/ml	2.26 ±1.21^a	107.52 ± 9.70^a	19.11 ± 2.14	23.75 ± 1.21

^aP ≤ 0.001 vs. G4.

Figure 1. Ultrastructural data for apoptosis of thymocytes. Control (A); chronic morphine treatment (B); chronic morphine plus L-NAME treatment (C). Bar = 5 μm.

Figure 2. Agarose gel electrophoresis of DNA isolated from rat thymocytes. Lane 1 – DNA; lanes 2 and 4 – Control; lane 3 – chronic morphine treatment; lane 5 – chronic treatment with morphine plus L-NAME; lane 6 – DNA degrading products.

Table 6. Densitometric evaluation of NADPH diaphorase positive staining in brain stem nuclei.

Group	Periaqueductal grеy matter	Dorsal raphe nucleus	Pedunculopontine tegmental nucleus	Laterodorsal tegmental nucleus
G1	165.6 ± 6.1^a (N = 2192)	175.4 ± 3.4^a (N = 797)	138.4 ± 3.2^a (N = 1694)	142.7 ± 4.1^a (N = 1019)
G2	120.0 ± 4.7^a (N = 1894)	143.5 ± 7.3^a (N = 907)	117.6 ± 4.1^a (N = 1290)	121.4 ± 3.9^a (N = 598)
G3	147.2 ± 4.6^a (N = 2321)	153.6 ± 7.7^a (N = 692)	128.1 ± 4.6^a (N = 1534)	127.3 ± 5.1^a (N = 656)
G4	214.7 ± 5.5 (N = 1708)	226.9 ± 5.5 (N = 859)	208.3 ± 5.7 (N = 1376)	213.7 ± 6.2 (N = 812)
G5	241.1 ± 5.8^a (N = 2259)	241.6 ± 7.2^a (N = 807)	239.5 ± 6.7^a (N = 1902)	253.3 ± 1.1^a (N = 207)
G6	238.4 ± 5.6^a,b (N = 2458)	237.6 ± 5.9^a,b (N = 758)	237.9 ± 5.1^a,b (N = 1718)	242.5 ± 8.4^a,b (N = 900)
G7	189.5 ± 12.8 (N = 2043)	193.3 ± 13.4 (N = 930)	189.4 ± 16.7 (N = 1490)	201.1 ± 17.9 (N = 991)
G8	215.8 ± 6.9 (N = 2025)	217.2 ± 5.4 (N = 420)	219.7 ± 9.3 (N = 1652)	210.4 ± 15.7 (N = 1186)

^aP ≤ 0.001 vs. G4; ^bP ≤ 0.001 vs. G2; (N) number of revealed NADPH positive neurons.