Evaluation of wound healing after colon anastomosis in morphine and methylphenidate addicted rats

Authors

Abstract

Aim There is limited data on how wound healing is affected in people with substance use disorders, who are more likely to experience trauma or require surgery for other reasons. This study aimed to evaluate the effects of morphine and amphetamine-derived methylphenidate dependence on wound healing in the anastomosis line and the anterior abdominal wall following colon anastomosis.
Materials and Methods A total of 24 rats were used in the study, including control, morphine, and methylphenidate groups, with eight rats in each group. In the study, 10 mg/kg morphine was administered subcutaneously twice daily for seven days, and 20 mg/kg methylphenidate was administered intraperitoneally for seven days to induce addiction. Then, all groups underwent anastomosis following sigmoid colon full-thickness transection, after which the abdomen was closed. On the seventh day after surgery, wound healing in the anastomosis line and the abdominal wall was evaluated using anastomosis burst pressure measurement, hydroxyproline levels, and histopathological examinations of samples taken from the anastomosis line and the anterior abdominal wall.
Results The study found that anastomosis burst pressure was significantly lower in the morphine group. No epithelialisation was observed on the anastomosis line in the morphine or methylphenidate groups. The formation of connective tissue was found to increase significantly in the methylphenidate group on the anastomosis line.
Discussion With these findings, it is thought that it may increase the risk of anastomosis leakage, especially in the early period, in cases of morphine and methylphenidate addiction.

Keywords

Introduction

Wound healing is one of the most important issues in our daily practice. This complex process can be influenced by various patient-related factors and the medications they are taking. Recently, due to the growing prevalence of drug addiction in our country, we have encountered more patients who are likely to have experienced trauma due to their violent tendencies and aggressive behaviour. It is important to note that there is very little literature on how to treat these patients or the effect of substance abuse on wound healing. The term ‘drug’ is currently defined as the general name given to chemicals that can be taken orally, intravenously, intranasally, or by inhalation, and which activate the brain’s reward system through various pharmacological mechanisms, producing feelings of pleasure and elation [1]. Substances are categorised into different classes, including alcohol, caffeine, cannabis, phencyclidine and other hallucinogens, inhalants, opiates, sedative-hypnotics and anxiolytics, stimulants (such as amphetamines and cocaine), nicotine, and other or unknown substances [2].
The relationship between substance use and violent behaviour is complex and suggestive, rather than definitive [3].
Similar increases have been observed in people taking psychotropic drugs, such as benzodiazepines and opiates. Therefore, the increased risk of trauma associated with the use of these substances may result in more injuries [4]. Some studies have found cocaine and opiates in the bodies of people who died in motor vehicle accidents or as a result of violence [5–8].
One study found cocaine in the system of 18.2% of people who died within 48 hours of being injured in a road traffic accident [6], while another study found cocaine in the system of 31.0% of homicide victims who died within 48 hours of being shot [7]. Similarly, one study found that 55,3% of homicide victims were dependent on substances (24,2% were dependent on cocaine, 26,9% on alcohol, opium (9.6%), cannabis (19.5%) [8]. This study investigated the impact of substance abuse on wound healing, evaluating the process at the anastomotic site and anterior abdominal wall following colonic anastomosis.

Materials and Methods

The study used 24 adult Wistar albino rats weighing between 250 g and 300 g. Rats were divided into three groups of 8 animals each: morphine, methylphenidate, and control. The rats were housed in individually ventilated cages (IVCs) at a temperature of 19–21°C, under a 12-hour light/dark cycle.
Creating Addiction
To induce morphine addiction in rats, a subcutaneous injection of morphine hydrochloride (Morphine HCl 10 mg/1 ml injectable solution, Galen, Istanbul, Turkey) was administered at a dose of 10 mg/kg every 12 hours for seven days [9]. It is noteworthy that rats that displayed aggressive movements during injections in the first few days became calmer after a few days. Accordingly, methylphenidate (Concerta® 36 mg controlled-release tablets, manufactured by Johnson & Johnson in New Brunswick, New Jersey, USA) was dissolved in saline and injected intraperitoneally once daily for five days in rats in the methylphenidate group. Rats that were not injected on day six were injected again on day seven at a dose of 20 mg/kg [10]. When preparing the drug for injection, the capsule was removed, and the long-release part was cut off and discarded. Surgical Procedure General anaesthesia was induced via the intraperitoneal administration of 5 mg/kg of xylazine (Rompun®, 2% solution, 25 mL vial, Bayer AG, Leverkusen, Germany) and 35 mg/kg of ketamine hydrochloride (Ketalar®, 500 mg/10 mL solution for injection, Pfizer, New York, USA). The abdomen was then entered via a median laparotomy through an approximately 3 cm incision. The sigmoid colon was located, and a full-thickness transection was performed while preserving the feeding vessels. A single-layer, end-to-end anastomosis was performed on the transected colon segment using eight (±1) 6/0 polypropylene sutures (Prolene®, Ethicon Inc., Somerville, New Jersey, USA) with a round needle.
Assessment of Recovery
Two animals in the methylphenidate group and one animal in each of the morphine and control groups died on the first and second days after surgery. On the seventh day after anesthesia, the anterior abdominal wall was excised with wide margins at the incision site of the rats that were sacrificed using a 55 mg/kg ketamine injection. Half of the tissue was placed in a 10% buffered formaldehyde solution (HistoPot®, Serosep Limited, Annacotty, County Limerick, Ireland) for histopathological examination. The other half was removed using a standard 3 mm diameter biopsy punch (Schuco Stiefel Biopsy Punch 3 mm, London, England) and placed in a 1.5 ml microcentrifuge tube (Eppendorf Tubes®, Hamburg, Germany), then stored at -80°C with saline added. The burst pressure of the anastomosis was measured using a manometer attached to an air sphygmomanometer. A 16-gauge Silastic catheter was carefully inserted through the animal’s anus. The intact bowel tissue proximal to the anastomotic line was then closed with a clamp. The animal was then sacrificed and placed in a bowl of water, after which the intestine was slowly inflated using the manometer. To prevent leakage from the anus, the anal tissue was clamped without obstructing the lumen. The pressure was recorded as the burst pressure at the moment when air bubbles first appeared, and a pressure drop was observed on the manometer, indicating compromised intestinal integrity. Then, a 5 cm segment of the colon, including the anastomosis line in the middle, was resected. One half of the anastomotic line was placed in a 10% buffered formaldehyde solution (HistoPot®, Serosep Limited, Annacotty, County Limerick, Ireland) for histological examination. The other half was removed using a 3 mm Schuco Stiefel biopsy punch for biochemical analysis, placed in an Eppendorf Tubes® 3810X microcentrifuge tube, and stored at -80°C after saline had been added. For histopathological examination, all materials from the anterior abdominal wall and the anastomotic line were collected. Routine tissue processing was performed, and paraffin blocks were prepared. Serial sections, each 3 microns thick, were stained with haematoxylin and eosin. The following were scored: epithelialization; polymorphonuclear leukocyte (PMN)/mononuclear leukocyte (MNL) ratio (indicative of the severity of inflammation); neovascularization; muscle layer; and connective tissue [11, 12] (Table 1; Figures 1, 2, 3, 4, and 5).
For the biochemical assay, standard-size frozen tissue samples from the anterior abdominal wall and anastomotic line were taken. These samples were stored at -80°C and then homogenized. Hydroxyproline was then determined in the supernatant of the homogenate (the liquid portion remaining after centrifugation and precipitation) using a solid-phase sandwich ELISA (enzyme-linked immunosorbent assay) with a commercial kit (Bioassay Technology Laboratory, Junijiang Inter. Bldg., Shanghai, China). The same samples were also assayed for protein content using the Bradford method, and the results were expressed as hydroxyproline per 1 mg of protein. Chitin, measuring range: 10–4000 ng/L; intra-assay CV: < 8%, and the between-assay CV: < 10%.
Statistical Analyses
The IBM SPSS Statistics 22 program (IBM SPSS, Türkiye) was used for the statistical analysis of the findings obtained in the study. During evaluation of the study data, the Shapiro–Wilk test was used to assess suitability for normal distribution; it was determined that the parameters did not follow a normal distribution. The Kruskal–Wallis test was used to compare quantitative data between groups, and the Mann–Whitney U test to identify the group responsible for the difference. The chi-square test and Fisher’s exact chi-square test were used to compare qualitative data. Significance was evaluated at the p < 0.05 level.
Ethical Approval
This study was approved by the Istanbul Mehmet Akif Ersoy Experimental Research, Development and Training Centre (IDEA) (Date: 2018-08-17, No: 2018/13).

Results

There is a statistically significant difference in the mean anastomotic burst pressure between the groups (p = 0.048; p < 0.05). Following pairwise comparisons to identify the source of significance, it was found that the mean anastomotic pressure of the morphine group was significantly lower than that of the control group (p = 0.033; p < 0.05). Although the mean anastomotic pressure of the morphine group was lower than that of the methylphenidate group, this difference was almost significant, but not quite (p = 0.055; p > 0.05).
There was no statistically significant difference between the mean anastomotic pressures of the control and methylphenidate groups (p = 0.755; p > 0.05) (Table 2). There was no statistically significant difference in hydroxyproline ELISA levels in the colonic anastomosis line between groups (p > 0.05) (Table 3). Similarly, there was no statistically significant difference in hydroxyproline ELISA levels in the anterior abdominal wall between the groups (p > 0.05).
There is a statistically significant difference in the level of epithelialization in the anastomosis line between the groups (p=0.003; p<0.05) (Table 4). This difference is due to the control group. While 28.6% of epileptic seizures in the control group were grade 0, 42.9% were grade 1, and 28.6% were grade 3, all epileptic seizures in the morphine and methylphenidate groups were grade 0 (p1 = 0.000, p2 = 0.000, p < 0.05). No epithelialisation was observed in addicts.
There was no statistically significant difference in PML/ MNL levels in the anastomotic line between groups (p>0.05) (Table 5). Similarly, there was no significant difference in PML/MNL levels in the anterior abdominal wall between the groups (p>0.05) (Table 5). Fisher’s Exact Test also showed no statistically significant difference between the groups in the level of neovascularisation in the anastomosis line (p > 0.05). Similarly, there was no statistically significant difference in the level of neovascularisation in the anterior abdominal wall (p > 0.05). However, there was a statistically significant difference between the groups in the amount of connective tissue at the anastomosis line (p < 0.05). This difference is due to the control group. All connective tissue in the control group was grade 1. In the morphine group, 42.9% were grade 0, 14.3% were grade 1, and 42.9% were grade 2 (p = 0.005, p < 0.05). In the methylphenidate group, none were grade 0, 16.7% were grade 1, and 83.3% were grade 2 (p = 0.005, p < 0.05). However, there was no statistically significant difference in connective tissue levels between the morphine and methylphenidate groups (p = 0.241; p > 0.05). There was no statistically significant difference in the amount of connective tissue in the anterior abdominal wall between the groups (p > 0.05).

Discussion

A significant proportion of morbidity and almost one-third of mortality following colorectal surgery is attributed to anastomotic leakage [13]. Studies involving substances that prevent epithelial migration have demonstrated that a lack of epithelialisation, particularly during the initial stages, can lead to anastomotic leakage due to an inability to fulfil its barrier function [14]. In our study, no epithelialisation was observed in the anastomosis line in the morphine and methylphenidate groups. Additionally, anastomotic burst pressures, which assess the strength of the anastomosis, were significantly lower in the morphine group. However, it is difficult to speculate on the mechanism by which morphine addiction causes these effects, as healing is a complex process involving many factors, and there is very little literature on wound healing in addicts. In fact, this is the first study to evaluate the healing of colonic anastomoses in people with addictions, and no other studies on this subject have been found in the literature. It is surprising that the effects of methylphenidate, a widely used drug especially among children, on wound healing, and the factors that should be considered before and after surgery in drug users, have not been studied. Pacifici et al. observed that, alongside the increase in serum heroin and morphine metabolites, the levels of cytokines such as IL-1β, IL-2, IL-10, TNF-α, TGF-β1, and IFN-γ also increased in serum. They demonstrated that a single injection of heroin and morphine increased the production of IL-1β, IL-2, TNF-α, and IFN-γ. After 24 hours, the production of these cytokines was inhibited, stimulating the release of anti-inflammatory cytokines such as IL-10 and TGF-β1. This suggests that this biphasic effect regulates the immune system and wound healing [15]. In a study investigating the cause of systemic fibrosis in morphine addicts, Wu et al. demonstrated that high doses of morphine increased the amount of connexin 43 (Cx43) and the secretion of adhesion molecules, such as connexin and alpha-smooth muscle actin, via TGF-β1 signalling. This resulted in an excessive increase in the transformation of fibroblasts into myofibroblasts, leading to impaired angiogenesis and delayed wound healing [16]. In our study, no significant differences in neovascularisation were observed in the anastomotic line or the anterior abdominal wall between morphine and methylphenidate addicts.
Alicea et al. demonstrated that stimulation of kappa opioid receptors inhibits the release of cytokines, such as IL-1, IL-6, and TNF-α, from macrophages [17]. Neudeck et al. revealed that stimulation of kappa opioid receptors reduces IL-8 secretion from intestinal epithelial cells [18], whereas stimulation of mu opioid receptors with endomorphin 1 increases IL-8 production [19]. In Iran, where rates of opiate addiction are high, Vahedian et al.’s study provides important information on the effects of morphine addiction on wound healing. The researchers found that the number of fibroblasts at the wound site increased on the seventh day after surgery in morphine-addicted rats compared to the control group [20]. In our study, morphine was found to make a statistically significant difference to connective tissue levels at the anastomotic line compared to the control group; however, no consistent data could be obtained regarding an increase or decrease. In our study, the low anastomotic burst pressures and lack of difference in hydroxyproline levels observed in the morphine group are consistent with studies showing that collagen strength and tensile strength generation are influenced by many factors. While determining the amount of collagen formed by hydroxyproline levels is important for providing a quantitative value when evaluating wound healing, studies have shown that mechanical methods, such as anastomotic burst pressure, are more valuable for evaluating the strength of formed tissue because collagen formation is affected by many factors [21].
Vahedian et al. found that the number of neutrophils at the wound site was lower in morphine-addicted rats than in the control group [20]. In our study, there was no significant difference in the PML/MNL ratio at the anastomotic line and the anterior abdominal wall. Vahedian et al. also found that angiogenesis decreased with morphine dependence. In our study, no significant difference was observed between groups with regard to neovascularisation at the anterior abdominal wall and anastomotic line. However, we found that epithelialisation at the anastomotic line was significantly lower in the morphine and methylphenidate groups.
No studies have evaluated the effects of methylphenidate on wound healing. Parrado et al. demonstrated that dopamine regulates inflammatory cell migration, remodelling, and angiogenesis by increasing the release of proinflammatory cytokines, such as IL-6 and IL-8, and metalloproteinases from keratinocytes and macrophages [22]. Conversely, Sarkar et al. concluded that dopamine inhibits angiogenesis [23]. In our study, the decreased epithelialisation in the anastomosis line in methylphenidate-addicted rats indicates an effect; however, the cause cannot be determined. The same effect is present in the morphine group.

Limitations

Our limiting factors were the small number of rats.

Conclusion

In morphine and methylphenidate addicts, no epithelialization was observed after colonic anastomosis. Anastomotic burst pressures were found to be lower in the morphine group. These findings suggest that morphine and methylphenidate dependence may increase the risk of anastomotic leakage, especially in the early period. In morphine and methylphenidate dependent patients, caution should be exercised when performing anastomosis in colon surgery, and the ostomy option may be safer in high-risk patients.

References

1. Göker Z, Üneri Ö, Dinç G, Güney E, Hekim Bozkurt Ö. Çocuk ve ergenlerde madde kullanım bozukluğuna yeni bir bakış: Dsm-5’in getirdikleri [A new perspective on substance use disorders in children and adolescents: what DSM-5 brings]. Turk J Child Adolesc Ment Health. 2015;22(2):131-9.
   PubMed Google Scholar
2. Güleç G, Köşger F, Eşsizoğlu A. DSM-5’te alkol ve madde kullanım bozuklukları [Alcohol and substance use disorders in DSM-5]. Curr Approaches Psychiatry. 2015;7(4):448-60.
   PubMed Google Scholar
3. Boles SM, Miotto K. Substance and violence: a review of the literature. Aggress Violent Behav. 2003;8(2):155-74.
   Article PubMed Google Scholar
4. Cordovilla-Guardia S, García-Jiménez C, Fernández-Mondéjar E, et al. Association between the detection of alcohol, illicit drugs and/or psychotropic medications/opioids in patients admitted due to trauma and trauma recidivism: A cohort study. PLoS One. 2018;13(9):e0203963.
   Article PubMed Google Scholar
5. Soderstrom CA, Dischinger PC, Kerns TJ, Kufera JA, Mitchell KA, Scalea TM. Epidemic increases in cocaine and opiate use by trauma center patients: documentation with a large clinical toxicology database. J Trauma. 2001;51(3):557-64.
   Article PubMed Google Scholar
6. Nordstrom DL, Yokoi-Shelton ML, Zosel A. Using multiple cause-of-death data to improve surveillance of drug-related mortality. J Public Health Manag Pract. 2013;19(5):402-11.
   Article PubMed Google Scholar
7. Tardiff AK, Wallace Z, Tracy M, Piper TM, Vlahov D, Galea S. Drug and alcohol use as determinants of New York City homicide trends from 1990 to 1998. J Forensic Sci. 2005;50(2):470-4.
   Article PubMed Google Scholar
8. Galea S, Ahern J, Tardiff K, Leon AC, Vlahov D. Drugs and firearm deaths in New York City, 1990-1998. J Urban Health. 2002;79(1):70-86.
   Article PubMed Google Scholar
9. Elahi-Mahani A, Heysieattalab S, Hosseinmardi N, Janahmadi M, Seyedaghamiri F, Khoshbouei H. Glial cells modulate hippocampal synaptic plasticity in morphine dependent rats. Brain Res Bull. 2018;140:97-106.
   Article PubMed Google Scholar
10. Wooters TE, Bardo MT. Nicotinic receptors differentially modulate the induction and expression of behavioral sensitization to methylphenidate in rats. Psychopharmacology (Berl). 2009;204(3):551-62.
    Article PubMed Google Scholar
11. Pehlivanlı F, Aydin O, Karaca G, et al. Healing of ischemic colon anastomosis in rats could be provided by administering dexpanthenol or coenzyme Q10. J Clin Med. 2018;7(7):161.
    Article PubMed Google Scholar
12. Kisli E, Ozdemir H, Kösem M, Sürer H, Ciftçi A, Kanter M. Effect of Ginkgo biloba extract (EGb 761) on the healing of left colonic anastomoses in rat. World J Surg. 2007;31(8):1652-7.
    PubMed Google Scholar
13. Jannasch O, Klinge T, Otto R, et al. Risk factors, short and long term outcome of anastomotic leaks in rectal cancer. Oncotarget. 2015;6(34):36884-93.
    PubMed Google Scholar
14. Rehn M, Krarup PM, Christensen LH, Seidelin JB, Ågren MS, Syk I. GM6001 increases anastomotic leakage following colonic obstruction possibly by ımpeding epithelialization. Surg Infect (Larchmt). 2015;16(6):702-8.
    Article PubMed Google Scholar
15. Pacifici R, di Carlo S, Bacosi A, Pichini S, Zuccaro P. Pharmacokinetics and cytokine production in heroin and morphine-treated mice. Int J Immunopharmacol. 2000;22(8):603-14.
    Article PubMed Google Scholar
16. Wu PC, Hsu WL, Chen CL, et al. Morphine induces fibroblast activation through up-regulation of connexin 43 expression: implication of fibrosis in wound healing. Int J Med Sci. 2018;15(9):875-82.
    Article PubMed Google Scholar
17. Alicea C, Belkowski S, Eisenstein TK, Adler MW, Rogers TJ. Inhibition of primary murine macrophage cytokine production in vitro following treatment with the kappa-opioid agonist U50,488H. J Neuroimmunol. 1996;64(1):83-90.
    PubMed Google Scholar
18. Neudeck BL, Loeb J, Buck J. Activation of the kappa-opioid receptor in Caco- 2 cells decreases interleukin-8 secretion. Eur J Pharmacol. 2003;467(1-3):81-4.
    PubMed Google Scholar
19. Neudeck BL, Loeb JM. Endomorphin-1 alters interleukin-8 secretion in Caco-2 cells via a receptor mediated process. Immunol Lett. 2002;84(3):217-21.
    PubMed Google Scholar
20. Vahedian J, Mirshekari TR, Nabavizadeh F. Effect of opium dependency on secondary intention wound healing in a rat model: an experimental study. Int Wound J. 2013;10(3):351-5.
    Article PubMed Google Scholar
21. Rygl M, Novotna J, Herget J, Skaba R, Snajdauf J. Parameters of healing in approximative intestinal anastomosis. Eur J Pediatr Surg. 2009;19(1):25-9.
    PubMed Google Scholar
22. Parrado AC, Salaverry LS, Mangone FM, et al. Differential response of dopamine mediated by β-adrenergic receptors in human keratinocytes and macrophages: potential implication in wound healing. Neuroimmunomodulation. 2017;24(4-5):282-91.
    Article PubMed Google Scholar
23. Sarkar C, Ganju RK, Pompili VJ, Chakroborty D. Enhanced peripheral dopamine impairs post-ischemic healing by suppressing angiotensin receptor type 1 expression in endothelial cells and inhibiting angiogenesis. Angiogenesis. 2017;20(1):97-107.
    Article PubMed Google Scholar

Declarations

Additional Information

Publisher’s Note
Bayrakol MP remains neutral with regard to jurisdictional and institutional claims.

Rights and Permissions

Creative Commons License

This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0). To view a copy of the license, visit https://creativecommons.org/licenses/by-nc/4.0/

View full license details →

About This Article