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Year : 2002  |  Volume : 56  |  Issue : 10  |  Page : 481-485

Standardisation of evoked response, threshold reaction time and morphine doses for analgesia in newborn nourished and undernourished suckling rats to noxious stimulus

1 Research Officer, Pharmacology, clinical Research Unit (H), Centre of Experimental Medicine and Surgery, India
2 Department of Pharmacology, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, India

Correspondence Address:
K P Singh
Research Officer (Pharmacology) Drug Standardisation Unit (H), Homoeopathic Pharmacopoea Laboratory, CGO Building Complex -1, Kamala Nehru Nagar, Ghaziabad - 201 002
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Source of Support: None, Conflict of Interest: None

PMID: 12712757

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How to cite this article:
Singh K P, Sanyal A K. Standardisation of evoked response, threshold reaction time and morphine doses for analgesia in newborn nourished and undernourished suckling rats to noxious stimulus. Indian J Med Sci 2002;56:481-5

How to cite this URL:
Singh K P, Sanyal A K. Standardisation of evoked response, threshold reaction time and morphine doses for analgesia in newborn nourished and undernourished suckling rats to noxious stimulus. Indian J Med Sci [serial online] 2002 [cited 2016 May 26];56:481-5. Available from:

Several reviews have appeared in the literature on procedures of quantitative analysis of the effectiveness of an analgesic drug in altering the sensation of pain. All these methods described the procedures for assessing the analgesia in adult animals. [1],[2],[3] For studying the analgesic effect of morphine in nourished and undernourished newborn suckling rats, it became imperative to adopt different criterias for evoked responses, threshold reaction time, and morphine doses over the already existed one for the "hot plate technique" used by earlier workers for adult animals, [4],[5],[6],[7],[8],[9] as newborn animals were highly sensitive.

 ¤ Materials and Methods Top

Albino rats (Fisher strain, 1 50-200g) ob­tained from IVRI, Izatnagar, Bareilly were fed Gold Mohur pellet diet (Lipton India Limited) ad-libitum. Adult females were bred in the departmental animal house for obtaining newborn suckling rats of a desired age. The day of delivery was re­corded as day 1 in order to record the actual age of the animal. Newborn suck­ling rats were undernourished accord­ing to the method of Mishra and Shankar. [10]

Hot plate analgesia apparatus (Techno) is a thermostatically controlled electri­cally heated plate surrounded by a trans­parent square box (22x22x15cm) with flexible lid on its top. Individual newborn suckling rat was placed gently on the hot plate through upper lid and the stop­watch was immediately pressed on. The animal was visualized carefully through the transparent wall for noting the characteristic cut off responses. As soon as the animal responded to noxious stimulus, the stopwatch was shut off and the animal taken away from the hot plate. The time interval between the threshold cut off response and on placing the animal on a hot plate, as measured in seconds by stopwatch, was taken as the threshold reaction time to induce pain to noxious stimulus.

Determination of threshold intensity: Earlier workers, while testing the anal­gesic effect of a drug in adult albino mice and rats, had maintained the tempera­ture of the "Eddy's hot plate" at 55° C. [4],[5],[6],[7],[8],[9] Nine days suckling rats, when placed on the hot plate maintained at 55°C, showed signs of discomfort at once. Hence, it became imperative to reduce the tem­perature of the hot plate to an extent that the animals responded to the threshold reaction time within the latent period of 2 to 8s (i.e.5±3 s). In the present study, the temperature of 55°C was found to be sufficient to elicit the characteristic cut off responses in newborn suckling rats.

Determination of cut off responses: In adult albino mice and rats, raising, kicking, dancing and licking of hind paws were considered as the "cut off responses" to the threshold reaction time. [4],[5],[6],[7],[8],[9] It was not practicable to follow the same criteria for 9 and 18 days suck­ling rats. In order to select the new re­producible characteristic "cut off responses", newborn suckling rats were placed on the hot plate maintained at a temperature of 50°C and visualized for reproducible characteristic threshold "cut off responses" elicited within the above mentioned latent period. The following "cut off responses" were selected for newborn suckling rats on different days of their age:

9 day-Running and turning on back; 18 days- Raising fore and hind paws alter­nately from the Floor. Approximately 75% of the animals urinated too, but it was not given any weightage in deciding the end point.

Determination of morphine doses: After taking two control threshold reac­tion times at 10 min intervals, morphine hydrochloride (Govt. Opium Factory, Ghazipur) in aqueous solution was ad­ministered subcutaneously to newborn suckling rats at different doses [Table 1] and [Table 2] in order to determine suitable doses which could prolong the control threshold reaction time above 60%, but not beyond 16 s. This period was con­sidered as maximum cut off time for morphine analgesia. The analgesic ef­fect of morphine was assessed at 15, 30, 45 and 60min intervals after its adminis­tration but the maximum increase in the latency of threshold reaction time was observed at 30 min interval, hence, it was taken for presenting the data in the text. The effective analgesic doses of mor­phine standardized in the present study for different age groups of newborn suck­ling rats were: a). 0.1 mg/kg s.c. for 9 days suckling rats; b). 1.0 mg/kg s.c. for 18 days suckling rats.

Statistical analysis: The mean value and standard error of mean of each group was calculated. The level of sig­nificance of the difference (probability of <0.05) between two groups was ana­lyzed by Student -'t' (paired) test.

 ¤ Results and Discussion Top

For adult rats and mice, the reported analgesic dose of morphine on Eddy's hot plate was <10mg/kg . [4],[5],[6],[7],[9] Morphine, when administered in doses of 5 mg/kg in 9 days suckling rats, not only failed to evoke any response to applied noxious stimulus but was also proved to be highly toxic. Hence, it became imperative to determine a suitable dose of morphine, which was safe, as well as, produced a reasonable degree of analgesia in different age groups of newborn suckling rats. The way of determining the analgesic dose of morphine in 9 and 18 days suckling rats is presented in [Table 1] and [Table 2]. The results showed that 9 and 18 days controlled nourished and undernourished suckling rats, when subjected to the noxious stimulus of the heat intensity of 50°C on a hot plate, responded with the characteristic reproducible "cutoff responses" within a reasonable latent period which varied from 2.73 ± 0.12 s to 3.53 ± 0.37s on different days of experimentation and the differences were not significant. In 9 days suckling rats, morphine in doses as low as 0.1 mg/kg increased the latency of the threshold reaction time significantly from its control value in both nourished (8.10 ±1.47s) and undernourished (5.23±0.88s) groups but in doses of 0.5 mg/kg, it was found to be highly lethal [Table 1]. On the other hand, 18 days suckling rats needed 10 times higher dose (1.0 mg/kg) of morphine for inducing a significant degree of analgesia [Table 2].

In the present study, 9 days suckling rats were found to be highly susceptible to morphine as compared to that of adult rats . [5] Kupferberg and Way [11] had reported 2-4 times higher levels of morphine in brain in 16 days rats as compared to that of 32 days old rats, after an intraperito­neal administration of equal doses of morphine, based on body weight of an animal. High membrane permeability/ fluidity of neuronal cells and incomplete development of blood brain barrier, [11],[12] low levels of plasma proteins and their binding capacity to drug molecules [13] and low levels of rug metabolizing enzymes [14] in newborn suckling rats might be the cause of such supersensitivity to mor­phine as more free morphine molecules were available to neuronal cells of the brain of newborn suckling rats. Subse­quent 10 times decreased in the sensi­tivity of developing rat brain on day 18th after birth to analgesic effect of mor­phine, as observed in present study [Table 2] could be explained by the de­velopment of the blood brain barrier and decrease in neuronal cells permeability in developing rats. [11],[15] Further studies in nourished and undernourished newborn suckling rats revealed that morphine an­algesia varied considerably on different days of undernourishment. [16]

 ¤ Summary Top

In the present study, the different criterias to assess the analgesic response of mor­phine in newborn suckling rats on "Eddy's hot plate" were modified. Initially, the intensity of applied heat stimulus to evoke the "cutoff response" was adjusted to 50°C. Different reproducible charac­teristic cut off responses (running and turning on back or raising fore and hind paws alternately) were selected for new­born suckling rats of varying age groups. The results revealed that newborn suck­ling rats are highly sensitive to morphine. The doses of morphine needed for pro­ducing a reasonable degree of analge­sia on body weight basis were 0.1 mg/ kg for 9 days old rats and 1.0 mg/kg for 18 days old rats. This procedure seems to be of great value in studying the anal­gesic effect of a drug in newborn suck­ling rats.

 ¤ References Top

1.Goetzl FR, Burril DY, IVY AC. A critical analysis of algesimetric methods with suggestions for a useful procedure Quart Bull Northw Univ Med School 1943; 17: 280-291.  Back to cited text no. 1    
2.Miller LC. A critique of analgesic testing methods. Ann N Y Acad Sci 1948,51:34-50.  Back to cited text no. 2    
3.Beecher HK. The measurement of pain: Prototype for the quantitative study of subjective responses. Pharmacol Rev 1957, 9: 59-209.  Back to cited text no. 3    
4.Eddy NB, Toucherry CF, Lieberman JE. Synthetic analgesic I: methadone isomers and derivatives. J Pharmacol Exp Therap 1950; 98: 121-137.  Back to cited text no. 4    
5.Eddy NB, Leimbach DG. Synthetic analgesic II: Dithienylbutenyl and dithienylbuttylamines. J Pharmacol Exp Therap 1953; 107: 385-393.  Back to cited text no. 5    
6.Janssen PAJ, Jageneau AH. A new series of potent analgesic: dextro 2, 2-diphenyl -3-methyl -4-morpholino butyrilpyrrolidine and related amides. Part I: Chemical structure and pharmacological activity. J Pharm Pharmacol 1957,9. - 381-400.  Back to cited text no. 6    
7.Alvarez-Fuentes J, Rojas-Corrales MO, Holgado MA, Caraballo I, Mico JA, Fernandez-Arevalo M. Preclinical study of an oral controlled release naltrexone complex. J Pharm Pharmacol 2000, 52: 659-663.  Back to cited text no. 7    
8.Capasso A, Loizzo A. Clonidine induced antimociception and locomotor hypoactivity are reduced by dexamethsone in mice. J Pharm Pharmacol 200 1; 53:351-360.  Back to cited text no. 8    
9.Alvarez-Fuentes J, Rojas-Corrales MO, Holgado MA, Sanchez-Lafuente C, Mico JA, Fernandez-Arevalo M. Effectiveness of repeated administration of a new oral naltrexone controlled-release system on morphine analgesia. J Pharrn Pharmacol2001; 53:1201-1205.  Back to cited text no. 9    
10.Mishra OP, Shanker R. Glycine transport in isolated cerebral tissue during malnourished and its response to neurotropic drugs. Indian J Biochem Biophys 1975, , 12.286-288.  Back to cited text no. 10    
11.Kupferberg HJ, Way El. Pharmacologic basis for the increased sensitivity of new born rat to morphine. J Pharmacol Exp Therap 1963; 141: 105-112.  Back to cited text no. 11    
12.Vanier MT, Holm M, Ohman R, Svennerholm L. Developmental profile of ganglioides in human ant rat brain. J Neurochem 1971; 18: 581-592.  Back to cited text no. 12  [PUBMED]  
13.Ehrnebo M, Agurell S, Jalling B, Boreus LO. Age differences in drug binding by plasma proteins. Eur J Clin Pharmacol 1971; 3: 189-193.  Back to cited text no. 13    
14.Dickerson JWT, Pao SK. Effect of pre-and post-natal maternal protein deficiency on free amino acids and amines of rat brain. Biol Neonat 1974; 25:114-124.  Back to cited text no. 14    
15.Long RA, Hruska F, Gesser HD, Hsia JC, Williams R. Membrane condensing effect of cholesterol and the role of its hydroxyl group. Biochem Biophys Res Commun 1970, - 41:321­328.  Back to cited text no. 15  [PUBMED]  [FULLTEXT]
16.Singh KP. Biochemical Pharmacology of Undernutrition in Rat Brain, Ph. D. Thesis, Banaras Hindu University, Varanasi., 1989.  Back to cited text no. 16    


  [Table 1], [Table 2]

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