Effect of Various Acupuncture Treatment Protocols upon Sepsis in Wistar Rats M. V. R. SCOGNAMILLO-SZABÓ,a G. H. BECHARA,a S. H. FERREIRA,b AND F. Q. CUNHAb
aFaculty
of Agricultural and Veterinary Sciences, São Paulo State University, Jaboticabal–SP, Brazil bFaculty
of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto–SP, Brazil
ABSTRACT: Sepsis is a syndrome characterized by infection and generalized inflammatory response that can lead to organ failure and death. In this study we standardize a model to investigate acupuncture’s effects upon sepsis. the objectives were to study the use of acupuncture in the infectious process and to formulate acupuncture’s treatment protocol for sepsis. The CLP (cecal ligation and puncture) model in rats was used to induce sepsis through bacterial entrance into the peritoneal cavity. An acupuncture treatment protocol that enhanced survival and reversed the neutrophil impairment migration toward the peritoneal cavity in rats with sepsis was achieved. It seems that acupuncture can be used for the treatment of experimental infectious processes. The effects of acupuncture and related mechanisms are discussed. KEYWORDS: acupuncture; sepsis; rats; neutrophil migration; cecal ligation and puncture
INTRODUCTION Acupuncture is the insertion of needles in cutaneous-specific locations of the body, known as acupoints, for the treatment or prevention of several diseases, including asthma, rhinitis, inflammatory bowel disease, and rheumatoid arthritis.1–5 Although this technique is increasingly used for the treatment of pain and other conditions, the rational basis underling its use remains unclear. Improved knowledge of the acupuncture therapeutic mechanism is essential to validating it since acupuncture is difficult to test under double-blind and placebo-controlled conditions.6–11 In fact, few reports concerning the effect of acupuncture on inflammatory/infectious models are available, although clinical trials claim the success of acupuncture in inflammatory disorders. Address for correspondence: M.V.R. Scognamillo-Szabó, Faculty of Agricultural and Veterinary Sciences, São Paulo State University, Jaboticabal–SP, Brazil. Voice: +55-16-3209-2662; fax: +55-16-3202-4275.
[email protected] Ann. N.Y. Acad. Sci. 1026: 251–256 (2004). © 2004 New York Academy of Sciences. doi: 10.1196/annals.1307.039 251
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Sepsis and septic shock are intense systemic inflammatory responses with multiple physiological and immunological abnormalities commonly caused by bacterial infection and possibly leading to organ failure and death.12 Neutrophil migration to the infectious focus is extremely important for the control of bacterial growth and consequently for the prevention of bacterial dissemination. The importance of this phenomenon to the evolution of sepsis has been demonstrated clearly in a cecal ligation and puncture (CLP) model. In lethal CLP, failure of neutrophil migration to the infectious focus is accompanied by an increased number of bacteria in the peritoneal fluid and blood and by a high mortality rate. Conversely, in nonlethal CLP, in which the impairment of neutrophil migration is not observed, the bacterial infection is restricted to the peritoneal cavity, and the animals exhibit increased survival.13–16 In this study we standardized a protocol of treatment to investigate the effects of acupuncture on neutrophil migration in sepsis. The cecal ligation and puncture (CLP) model was used in rats to induce the entrance of bacteria into the peritoneal cavity, leading to sepsis.
MATERIALS AND METHODS Induction of Sepsis Male Wistar rats (weight 230–250 g) were used throughout the study. Sepsis was induced by cecal ligation and puncture (CLP), as described elsewhere.13 Briefly, rats were anesthetized with 2.5% tribromoethanol, and the cecum was exposed and ligated below the ileocecal junction, without causing bowel obstruction, and punctured 4 or 20 times with a 16G gauge needle. Animals given 4 or 20 punctures show 100 or 0% survival, respectively; we called these nonlethal (NL) and lethal (L) CLP, respectively. Experimental Design Animals that underwent CLP were submitted to two different protocols of manual acupuncture (test groups). Six hours later, treatment test and control (nontreated) groups were analyzed for neutrophil migration and bacteria count in the peritoneal cavity. Negative control groups consisted of naive animals. All experiments were performed in the morning between 8:00 A.M. and 11:00 A.M. to avoid circadian interference.17,18 Treatments Manual acupuncture was performed immediately after CLP. Detailed descriptions of treatment protocols are shown in TABLES 1 and 2. The acupoints were selected based on traditional treatment of fever-related diseases.19 For acupuncture sessions, animals were lightly immobilized using hands to minimize stress. The nontreated control group was also lightly immobilized using the same method. Acupuncture points were identified by one of the authors (M.V.R.S.S), an experienced acupuncturist.
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TABLE 1. Acupuncture treatment protocol 1 used in rats with CLP-induced sepsis Time
Acupoint
Acupoint Localization
Zero
S 36
2h
GV 01
Midline between anus and tail
4h
GV 01
Midline between anus and tail
Below the cranial crest of the tibia, in the belly of the tibialis cranialis muscle
TABLE 2. Acupuncture treatment protocol 2 used in rats with CLP-induced sepsis Time
Acupoint
Acupoint Localization
Zero
B 25
Between the apical ends of the transverse process of L5-L6 vertebrae
1:30 h 4:30 h
6h
GV 01
Midline between anus and tail
GV 03
Lumbosacral space
GV 01
Midline between anus and tail
GV 14
Space between C7 and T1 vertebrae
Liv 02
In the distal third of the depression between metatarsal I and II
Er Jien
On the convex surface of the tip of the ear
GV 14
Space between C7 and T1 vertebrae
LI 11
In the depression between the dorso-lateral condyle of humerus and the processes anconaeus
10 h
GV 01
Lumbosacral space
14 h
GV 01
Lumbosacral space
Neutrophil Migration and Number of Bacteria in Peritoneal Cavities Six hours after CLP the animals were killed in an ether chamber, and the peritoneal cavity was washed by injecting 10 mL of sterile PBS containing 1 mM EDTA. Aliquots of serial log dilutions of the peritoneal lavage fluid were plated on MillerHinton agar dishes. Colony-forming units (CFU) were counted after overnight incubation at 37°C, and the results were expressed as the number of CFU per cavity. Total leukocyte counts were made in a cell counter, and differential cell counts were made on cytocentrifuge slides stained by the May-Grünwald-Giemsa method (Rosenfeld). The results are expressed as the number of neutrophils per cavity. Statistical Analyses Neutrophil and bacteria counts are reported as mean ± SE. The means between different treatments were compared by analysis of variance. If significance was determined, individual comparisons were subsequently tested with the Bonferroni test for unpaired values. Statistical significance was set at P < .05.
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FIGURE 1. Effect of acupuncture treatment protocols on neutrophil migration and bacterial counts in the peritoneal cavities of rats 6 h after nonlethal (NL) and lethal (L) cecal ligation and puncture (CLP). Neutrophil migration and quantification of the amount of bacteria were also determined in naive rats. Results are expressed as mean ± SE of neutrophils per cavity and as mean CFU per cavity and are representative of two independent experiments. The number of animals in the different experimental groups are indicated below the bars. #P < .05 and *P < .001 compared with NL-CLP (analyses of variance, followed by the Bonferroni test).
RESULTS The number of bacteria present in the peritoneal cavities of L-CLP rats was 2.5fold higher than that observed in NL-CLP. Despite this, the L-CLP group presented failure of neutrophil migration into peritoneal cavities, compared with NL-CLP. The impairment of neutrophil migration observed in L-CLP was partially reverted by acupuncture (FIG. 1A and C), and, as a consequence, the number of bacteria present in the peritoneal cavities of acupunctured rats was lower compared with L-CLP nontreated animals (FIG. 1B and D). The reestablishment of neutrophil migration in L-CLP was more evident with protocol 1.
DISCUSSION During sepsis several physiopathological events are clearly associated with the presence of the inflammatory mediators in the circulation, which induces accumula-
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tion and activation of leukocytes, in important organs, such as the lung. Nevertheless, inhibition of neutrophil migration to infection focus is observed and often associated with uncontrolled bacterial growth and subsequent bacteremia, which may then lead to increased lethality.20 The relationship found between reduced neutrophil chemotaxis and poor prognosis in human subjects suggests that restoration of the neutrophil chemotatic function could be an appropriate strategy in the septic patient.14 The high level of cytokines and nitric oxide in the circulation seems to mediate this event. So, pharmacological approaches that reduced the production of systemic cytokines and nitric oxide reestablished the neutrophil migration failure and consequently controlled the infection focus.14–16 In the present study we observed that acupuncture partially reestablished the neutrophil migration and reduced the number of bacteria in the infectious focus. The effect of acupuncture on the production of systemic cytokines and nitric oxide is under investigation. In this regard, there is evidence in the literature indicating that acupuncture treatment reduces the intensity of the inflammatory process observed in experimental and human inflammatory diseases, including arthritis, epicondylitis, complex regional pain syndrome type 1, vasculitis, and reduces the production of the inflammatory mediators.3–5,21–27
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