INTRODUCTION

The newer quinolones, the fluoroquinolones are represented by Cipro-floxacin, Norfloxacin, Ofloxacin and Tamofloxacin. These agents represent an improvement over their quinolone counterparts in many ways including a wider spectrum of anti-microbial activity, improved pharmacokinetic properties, and clinical efficacy against wider range of diseases.¹ Ciprofloxacin is one of the more active in this calss that possesses an extended spectrum of activity², has good bioavailability 80-95% and distributed widely in body fluids and tissues.³ It has sufficiently long serum half-life to allow twice daily dosing.⁴

Quinolones cause cartilage damage deterioration in young animals and are not recommended for use in children less than 18 years or pregnant or nursing women.⁵ After initiation of ciprofloxacin in clinical trials in the early 1980s it has been administered to children despite restrictions. The first report from a child treated with ciprofloxacin was in May 1983.⁶ Since that time ciprofloxacin has been continuously used in children and adolescent when conventional therapy failed or was not available.

Studies on the effects of ciprofloxacin on bone growth have been carried out, but any effect on skeletal differentiation has not been reported so far. This study was done to evaluate the effect of ciprofloxacin administration on differentiation of long bones in extremities of postnatal juvenile laboratory Wistar albino rats.

MATERIAL AND METHODS

In this study 30 spontaneously ovulating female & 15 fertile male Wistar albino rats, were 10-12 weeks of age, when taken from the Animal House of BMSI, JPMC, Karachi., The female rats were mated with fertile males of the same strain, according to the method described by Rough⁷ allowing one male rat with two female rats in one cage. On next morning the female rats were examined for signs of mating in the form of blood stained vagina or a vaginal plug (a mucoid greenish white material). Presence of any one of these signs was considered as day-1 of pregnancy⁸. The gestation period of the albino rat is usually between 21 and 23 days.⁹

Thirty pregnant albino rats were allowed to deliver their pups. Randomly selected 140 pups were divided into two groups, i.e. A and B, each comprising 70 animals. Sex of these offspring was omitted.

Group-A (experimental, n=70) pups were given injection Ciprofloxacin (developed in Bayer Research Laboratories, AG, Germany) at a dose of 20 mg/ kg body weight¹⁰ (0.12 mg in 0.1 ml) intraperitoneally twice daily for 14 days from day-1 after birth.

Group-B (control, n=70) pups were given normal saline in equal volume (0.1 ml)¹¹ intraperitoneally twice daily for 14 days from day-1 after birth.

Five specimens were then randomly selected for the study from each group for each day mentioned (total 70 pups from group experimental which received Ciprofloxacin and total 70 pups from control group which received Normal saline). Pups were then killed by deep ether anaesthesia and fixed in 80% alcohol after removing their skin and viscera. Pups were then bulk stained in Alizarin red and Alcian blue, cleared in 4% K(OH) revealing ossification centers and were stored in glycerin by the method as described by patton & Kafman.¹²

Left fore and hind limbs were separated from axial skeleton at their joints and viewed by magnification x4 under stereomicroscope. The presence or otherwise of secondary ossification centers in humerus and femur at their proximal and distal ends were observed and recorded. Care was taken to note the measurement of growth plate and its different zones. The said bones were observed throughout the experimental period (day-1 to day-14 postnatally). Even after the detection of primary ossification centers to see appearance of any additional ossification center in shaft or end of these long bones. The mean value of the time of first appearance of secondary center in experimental and control bone is given as mean±SEM. Student ‘t’ test was employed to determine the statistical significance of the results as described in ‘introduction of medical statistics’.¹³

RESULTS

The mean time of appearance of secondary ossification centers in major skeletal components of both extremities in experimental and control animals are given in table and shown in figures-1, 2 and 3.

Table-1: Comparison of Time (day) and Appearance of Secondary Ossification Centers in postnatal Experimental animals (group-A) and Control (group-B) at Proximal and Distal ends of Long bones: Left Humerus and Left Femur in Juvenile Wistar Albino rat pups

Bone		Experimental Animals (n=70)	Control (n=70)	Time Delay
Humerus	PE	9.4 ± 0.2*	7.0 ± 00	2.4 ± 0.2
Humerus	DE	9.4 ± 0.2*	7.0 ± 00	2.4 ± 0.2
Femur	PE	16.4 ± 0.2*	14.0 ± 00	2.4 ± 0.2
Femur	DE	9.2 ± 0.2*	7.0 ± 00	2.2 ± 0.2

Key:PE = Proximal end; DE = Distal end; n = Total number of animals;

*P<0.001 (highly significant increase)

Figure-1: Photograph of postnatal treated pups whole skeleton with double staining technique, i.e. Alizarin red stained bone and Alcian blue stained cartilage showing comparison of appearance of secondary ossification centers at proximal and distal ends of long bones between experimental [group-A (left)] and control [group-B (right)] animals used in this study.

Figure-2: Photograph of left fore limb (Humerus bone) with double staining technique, i.e. Alizarin red stained bone and Alcian blue stained cartilage showing comparison in presence of secondary ossification centers at proximal and distal ends between experimental [group-A (left)] and control [group-B (right)] animals used in this study.

Key: L = Left; HL = Hind limb

Figure-3: Photograph of left hind limb (Femur bone) with double staining technique, i.e. Alizarin red stained bone and Alcian blue stained cartilage showing comparison in presence of secondary ossification centers at proximal and distal ends between experimental [group-A (left)] and control [group-B (right)] animals used in this study.

Key: L = Left, FL = Fore limb

LEFT HUMERUS

In control animals, the secondary centers of ossification at proximal and distal ends were present on seventh postnatal day in all specimens observed (5 specimens of each bone). No additional ossification center was observed in any of the bones mentioned during the rest of study period.

In experimental animals, the secondary ossification centers at proximal and distal ends were seen to be present on ninth postnatal day in 3 specimens and on 10^th postnatal day in 2 specimens (average 9.4 days with delay of 2.4 days).

The mean epiphyseal growth plate thickness in control animals was 131.65+0.63 mm, while ciprofloxacin treated animals was 117.60 + 1.05 mm. the difference was found to be 14.05+0.37mm.

LEFT FEMUR

In control animals the secondary ossification centers were present at proximal end on 14^th day after birth in all specimens observed (5 specimens of each bone) while at distal end were present on 7^th postnatal day in all specimens (5 specimens of each bone). Any additional postnatal ossification center was not observed in any of these bones during rest of study.

In experimental animals the secondary ossification centers at proximal end was seen to be present on 16^th postnatal day in 3 specimens and on 17^th postnatal day in 2 specimens (average 16.4 days with delay of 2.4 days) while at the distal end the secondary ossification centers were present on 9^th postnatal day in 4 specimens and on 10^th postnatal day in one specimen (average 9.2 days with delay of 2.2 days).

The mean epiphyseal growth plate thickness in control animals was 139.65+0.39mm while in ciprofloxacin treated animals was 133.05+1.6mm. The difference was found to be 6.6+1.21mm.

DISCUSSION

Appearance of ossification center is the early indication of skeletal differentiation. The effect of ciprofloxacin treatment on skeletal differentiation was therefore studied by determining the time of first appearance of secondary representative ossification center in post-natal humerus and femur in experimental and control animal.

In control animals the secondary centers of ossification in humerus bone at proximal and distal ends appeared at 7^th post-natal day and in femora secondary center appeared at proximal end on 14^th post-natal day, while at distal end appeared on 7^th post-natal day in all specimens observed. Our observation is similar to the findings of Patton and Kaufman¹².

Ciprofloxacin delayed the appearance of secondary ossification centers in humerus at proximal and distal ends by 2.4±0.2 days as compared to control animals. This delay was statistically highly significant (P<0.001), whereas, ciprofloxacin delayed the appearance of secondary ossification centers in femora at proximal and distal ends by 2.4±0.2 days and 2.2±0.2 days respectively as compared to control animals. The delay averaged 2.4±0.2 days in fore limb for both ends and 2.4±0.2 days at proximal end and 2.2±0.2 days at distal end in hind limb bones. This concluded that fore limb and hind limb bones were affected by the adverse effect of ciprofloxacin.

In this study the delay in appearance of ossification centers by ciprofloxacin is attributed to tissue accumulation of fluoride which presumabily delayed that calcification process¹⁴ by inhibitory action on hydroxyappetite depostion¹⁵. The exact histo-chemical mechanism by which ciprofloxacin distrubs the ossification has not been investigated. Our observation is in consistance with the findings of Stahlmann¹⁶ who stated that in animals during early post-natal developmental the epiphyseal growth plate can be damaged. However he neither specified the type of growth plate damage nor clarified the form of the irreversible bone damage. Irreversible bone damage could be either in the form of retarded formation or abnormal formation.

Our study suggests that the growth plate damage is due to chondrocyte proliferation depression as shown by diminuation of proliferative zone of the epiphyseal growth plate. Arora stated that repeated fluoroquinolone administration causes floride accumulation in the bones particularly affecting the linear growth¹⁴.

Linear growth of long bones is through endochondral ossification¹⁷. This means that linear bone growth may be adversely affected primarily by cartilage growth retardation or secondarily by delay in conversion of the cartilaginous model into the newly formed bone. Since according to Arora floride is deposited into the bone, the bone growth appears to be retarded by the conversion into the newly laid bone. However, our finding of diminuation of the proliferation zone of cartilage in pups favours the idea that even if the bone growth is not entirely affected by the cartilage proliferation retardation, it must be the main determinant factor rather than floride deposition in bone.

In pups fusion of secondary ossification centers of long bones at age 6 weeks¹⁸. However, in humans the fusion of the secondary ossification centers of long bones occurs at 18 to 25 years. Considering epiphyseal delay of 2.4+0.2 days in pups in relation to 8 weeks age at skeletal maturation. The maturation to delay in maturation duration is 16.8:1. From our study model extrapolated value of delay in maturation in humans would be 478 days. However the ciprofloxacin injection period in pups was 14 days which is 1:3 ratio to the total maturation period.

Therefore if the ciprofloxacin is to be injected in the same ratio to the human skeletal maturation period it would be 7:21 (years). If the ciprofloxacin injection is for 7 years than delay maturation would be 478 days, but in reality ciprofloxacin injection in human children is upto 3 months. That will cause delay of 1:28 days. 478 ciprofloaxacin days delay in human divided by 2.4 ciprofloacin delay in pups would be 199 days ratio. Therefore 2677 days divided by 90 days, 30 times less period of ciprofloxacin injection in human therefore 2.4 days ¸ 30 = 0.08 days, delay would occur in humans if the adverse effect of ciprofloxacin is of similar magnitude. This delay is about 2 hours, since skeletal maturation range in humans varies from 18 to 25 years in which a difference of 2 hour delay is very highly statistically insignificant.

Therefore we consider the ciprofloxacin is very safe as far as its action on delayed epiphyseal closure is concerned. The time of appearance of secondary epiphysis is inversely related to the epiphyseal closure. Therefore the extrapolated values of delay in animals need to be confused in humans.

Conclusion

Since ciprofloxacin significantly inhibited the ossification process 2.4±0.2 days (delayed P<0.001), as compared with control. The ciprofloxacin administration post-natally presents a risk to skeletal differentiation and therefore to its growth upto the age six weeks in abino rat pups.

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