Honokiol for the Treatment of Neonatal Pain and Prevention of Consequent Neurobehavioral Disorders

Abstract

This study examined the short- and long-term neuroprotective and analgesic activity of honokiol (a naturally occurring lignan isolated from Magnolia) on developing brains in neonates exposed to inflammatory pain, known to cause neuronal cell death. Postnatal day 4 (P4) neonatal rat pups were subjected to intraplantar formalin injection to four paws as a model of severe neonatal pain. Intraperitoneal honokiol (10 mg/kg) or corn oil vehicle control was administered 1 h prior to formalin insult, and animals were maintained on honokiol through postnatal day 21 (P21). Behavioral tests for stress and pain were performed after the painful insult, followed by morphological examinations of the brain sections at P7 and P21. Honokiol significantly attenuated acute pain responses 30 min following formalin insult and decreased chronic thermal hyperalgesia later in life. Honokiol-treated rats performed better on tests of exploratory behavior and performed significantly better in tests of memory. Honokiol treatment normalized hippocampal and thalamic c-Fos and hippocampal alveus substance P receptor expression relative to controls at P21. Together, these findings support that (1) neonatal pain experiences predispose rats to the development of chronic behavioral changes and (2) honokiol prevents and reduces both acute and chronic pathological pain-induced deteriorations in neonatal rats.

Figure 1

Honokiol attenuates formalin-induced acute pain responses in neonatal rats. The response to inflammatory pain was tested in the “paw-licking test”, which included evaluation of all recuperation behaviors (licking, flinching, and rolling) in postnatal day 7 (P7) pups. Honokiol (10 mg/kg, ip) was dissolved in corn oil. Corn oil alone was tested as the vehicle control for honokiol treatment. Formalin (5%) sc injection is an established inflammatory pain model; saline injection was used as the control for the formalin insult. Honokiol or corn oil was administered 1 h before either formalin or saline injection into the left forepaw. (A) Formalin injection triggered recuperation behaviors including marked and prolonged licking activity of the injected paw at 5 min and again at 30 min after the formalin insult. (B) Saline injection did not cause significant increases in recuperation behaviors in either corn oil or honokiol pretreated rats. (C) Although there was no significant difference 5 min after the painful stimulus between rats in honokiol + formalin vs corn oil + formalin groups, honokiol treatment significantly reduced the licking duration at 30 min after formalin injection. Results are presented as the mean ± SEM (n = 8 animals per group). *p < 0.05 between corn oil + formalin vs honokiol + formalin groups at 30 min. p < 0.05 for corn oil + saline vs corn oil + formalin at 5, 30–35, and 55 min; honokiol + saline vs corn oil + formalin at 25–35 and 50–55 min; corn oil + saline vs honokiol + formalin at 5 min also. There was no statistically significant difference among the other groups and times analyzed.

Figure 2

Inflammatory pain induces chronic enhancement of pain sensation, attenuated by honokiol. Hot plate testing for thermal hyperalgesia was performed in P16/P17 rats subjected to control or formalin injection from P4 to P6. Honokiol or corn oil vehicle control was administered prior to formalin injection daily during the 3-day insult. Saline injection was used as a noninflammatory pain control for formalin. Rats in honokiol and corn oil groups were continuously treated with honokiol and vehicle control, respectively, every other day until P21 for the hot plate test. In saline injection control rats, the response latency to jumping on the hot surface (55 ± 2 °C) was similar between corn oil and honokiol groups. The latency of response, however, was noticeably shortened in rats of the formalin/corn oil group. Honokiol treatment largely prevented the development of thermal hyperalgesia compared to corn oil controls. Average 3 trials per animal. Results are presented as the mean ± SEM (n = 11, 10, 23, 22 in corn oil + saline, honokiol + saline, corn oil + formalin, honokiol + formalin groups, respectively). *p < 0.0001 for honokiol + formalin vs corn oil + formalin rats. p < 0.001 for honokiol + saline vs corn oil + formalin rats. No other groups showed statistically significant differences.

Figure 3

Honokiol prevents behavioral abnormalities resulting from early pain experiences. Two weeks after the neonatal inflammatory pain insult or control procedure, P20–21 rats were tested for chronic changes in their behavior activities. (A) In the defensive withdrawal test, rats that received formalin inflammatory pain preferred to hide inside the dark shelter, while rats in the formalin and honokiol group were more likely to leave the shelter of darkness for exploration outside the shelter. Results are presented as the mean ± SEM (n = 6, 7, 14, and 14 for corn oil + saline, honokiol + saline, corn oil + formalin, and honokiol + formalin groups, respectively). *p < 0.05 for honokiol + formalin vs corn oil + formalin and for honokiol + saline vs corn oil + formalin. p = 0.1619 for corn oil + saline vs honokiol + saline, indicating corn oil or honokiol alone did not change the defensive withdrawal behavior in normal saline control animals unexposed to formalin injury. (B) In novel object recognition testing, rats were exposed to a novel object after familiarization with the original object. The discrimination ratio (time spent with novel object vs time spent with original object) decreased in rats subjected to early inflammatory pain. Honokiol treatment prevented this functional deficit, and the discrimination ratio for honokiol + formalin treated rats was significantly increased compared to corn oil + formalin controls. Results are presented as the mean ± SEM (n = 7, 7, 12, and 12 for corn oil + saline, honokiol + saline, corn oil + formalin, and honokiol + formalin groups, respectively). *p < 0.05 for honokiol + formalin vs corn oil + formalin groups. No other groups showed statistically significant differences.

Figure 4

Honokiol attenuates pain-related upregulation of c-Fos expression in the brain. (A) c-Fos expression as an early marker of chronic pain was measured using immunohistochemical staining (dark brown) in brain sections from P7 rats after the 3-day formalin or control treatment (20× field). (B and C) Quantified data of c-Fos expression in the hippocampus (B) and thalamus (C). Results are presented as the mean ± SEM (n = 6, 6, 7, 8 animals per corn oil + saline, honokiol + saline, corn oil + formalin, and honokiol + formalin groups, respectively, for both hippocampal and thalamic sections). Formalin-induced inflammatory pain markedly increased c-Fos expression in the hippocampus. Honokiol largely prevented this increase of the chronic pain markers in formalin-injured animals (*p = 0.0122 for honokiol + formalin vs corn oil + formalin; p < 0.05 for corn oil + saline and honokiol + saline groups vs corn oil + formalin). The honokiol effect in the thalamus was not significant for honokiol + formalin vs corn oil + formalin groups (p = 0.0531). p < 0.05 for corn oil + formalin vs honokiol + saline groups. There was no difference in c-Fos expression within the saline groups or other groups in either the hippocampus or thalamus.

Figure 5

Inflammatory pain results in chronic downregulation of substance P receptor levels, attenuated by honokiol. In P21 rats, immunohistochemical staining with the substance P receptor antibody was used to detect the long-term effect of inflammatory pain on this pain-related receptor in the hippocampus. (A to C) The expression of substance P receptor in the brain was decreased 2 weeks after formalin insult. Honokiol treatment noticeably maintained the substance P receptor expression in the hippocampus. (D) Quantified data of the number of substance P receptor positive cells in the hippocampus. Saline or honokiol alone did not alter the expression level. There was a marked reduction in substance P receptor level 2 weeks after the 3-day inflammatory pain protocol. Honokiol treatment normalized the expression of substance P in the alveus of formalin-injured rats. Results are presented as the mean ± SEM (n = 7, 3, 6, 7 rats in corn oil + saline, honokiol + saline, corn oil + formalin, and honokiol + formalin groups, respectively). *p = 0.0018 for honokiol + formalin vs corn oil + formalin rats. p < 0.01 for corn oil + saline vs corn oil + formalin rats. There was no statistically significant difference between other groups analyzed.