Date Published: August 27, 2019
Publisher: Public Library of Science
Author(s): Masahiro Nakahashi, Mariko Esumi, Yasuaki Tokuhashi, Hee-Jeong Im Sampen.
Although low-back pain is considered to be associated with cigarette smoking, the influence of cigarette smoking on the intervertebral discs (IVD) has not been confirmed. We established a rat model of passive cigarette smoking-induced IVD degeneration, and investigated the cytohistological changes in the IVD and the accompanying changes in gene expression. IVD from rats exposed to 8 weeks of passive cigarette smoking were stained with Elastica van Gieson, and exhibited marked destruction of the supportive structure of the reticular matrix in the nucleus pulposus (NP). Positive signals on safranin O, alcian blue, type II collagen and aggrecan staining were decreased in the destroyed structure. Safranin O and type II collagen signals were also decreased in the cartilage end-plate (CEP) after 4- and 8-weeks of cigarette smoking. In the CEP, the potential for apoptosis was increased significantly, as demonstrated by staining for single-strand DNA. However, there were no signs of apoptosis in the NP or annulus fibrosus cells. Based on these findings, we hypothesized that passive cigarette smoking-induced stress stimuli first affect the CEP through blood flow due to the histological proximity, thereby stimulating chondrocyte apoptosis and reduction of the extracellular matrix (ECM). This leads to reduction of the ECM in the NP, destroying the NP matrix, which can then progress to IVD degeneration.
Low-back pain is a highly prevalent disease, and a major problem for performing activities of daily living and health economics. Many studies have reported that cigarette smoking is a risk factor for low-back pain; i.e., 70% of persons with low-back pain are cigarette smokers, and the frequency, amount and duration of cigarette smoking correlate with the incidence of low-back pain [1–9]. In contrast, smoking cessation has been reported to improve patient-reported pain and is related to increased fusion rates [10, 11]. Moreover, cigarette smoking is also associated with degenerative disc disease, including middle-aged disc herniation [12–15]. Cigarette smoking leads to the formation of carboxy-hemoglobin , vasoconstriction  and arteriosclerosis [16, 17], and thus decreases oxygen transport and blood flow [16, 17]. These events are considered to lead to malnutrition of the intervertebral discs (IVD) and promote IVD degeneration. Studies using animal models and in vitro culturing of disc cells suggested that nicotine and tobacco smoke exposure induces degenerative changes in the spine [18–22]. Although there have been significant advances in our understanding of the biology underlying IVD degeneration  , the molecular mechanisms underlying the IVD changes induced by cigarette smoking remain to be elucidated.
Using this model, we previously observed that rat body weight gain was suppressed by passive cigarette smoking (S5 Fig). A similar effect was observed in rats allowed to self-administer nicotine, independent of food intake, which corresponded to 15 to 60 micrograms/kg/infusion nicotine . We also measured blood nicotine levels in our previous study: 36.5 to 124.8 ng/ml (mean: 72.1 ng/ml) . This concentration range corresponds to 10 to 70 ng/ml (mean: 33 ng/ml) reported for 330 human smokers who smoked 20.7 cigarettes/day on average [25, 33]. Therefore, this model is comparable to humans who are smoking 20 to 40 cigarettes/day. Under these exposure conditions, the NP architecture was destroyed by passive cigarette smoking and the supporting structure composed of the ECM had degenerated based on EVG staining. This supportive structure comprised type II collagen and PG, which were both destroyed by passive cigarette smoking. Type II collagen and aggrecan were decreased at the protein level, but this was not supported by quantitative mRNA analysis. Although there were no increases of Mmp13 or Mmp3 mRNA in this study, Adamts4 expression was slightly attenuated in the cigarette smoking groups. Activation of other degrading enzymes may have been involved in the decrease of these matrix proteins. Recently, Ngo et al. demonstrated that ADAMTS5 is the primary aggrecanase mediating smoking-induced IVD degeneration in mouse models of chronic tobacco smoking using ADAMTS5-deficient mice . Thus, ADAMTS5 may also be the enzyme responsible for the degradation in the current study. Wang et al. reported marked loss of disc matrix in a mouse cigarette smoking model using direct smoke inhalation . As their model utilized direct vs. passive inhalation, the smoke conditions were more severe than those used in our study, and the degradation of aggrecan, and reduced synthesis of PG and collagen were also demonstrated. Thus, these findings support the conclusion that tobacco smoke alone is sufficient to affect peripheral tissues and lead to IVD degeneration.