versão impressa ISSN 0004-2803
Arq. Gastroenterol. vol.49 no.1 São Paulo jan./mar. 2012
Lesões gástricas induzidas pelo ruído: um estudo por microscopia ótica e eletrônica da parede gástrica do rato exposta a ruído de baixa frequência
Jorge FonsecaI; José Martins-dos-SantosI; Pedro OliveiraI; Nuno LaranjeiraI; Artur AguasII; Nuno Castelo-BrancoIII
IInstituto Superior de Ciências da Saúde Egas Moniz (CiiEM), Almada, Portugal
IIInstituto de Ciências Biomédicas Abel Salazar - Universidade do Porto (UMIB), Porto, Portugal
IIICenter for Human Performance, Alverca, Portugal
CONTEXT: Only a few studies evaluated the digestive alterations caused by low frequency noise (LFN) and most focused only on mucosal alterations.
OBJECTIVES: To investigate the morphological injury of LFN-exposed gastric wall, beyond the epithelial layer.
METHODS: Wistar rats were exposed to low frequency noise (LFN), during increasing periods, 1 to 13 weeks. A control group was kept in silence. Gastric specimens were studied using: (i) light microscopy with hematoxylin-eosin and immunostaining for collagens; (ii) transmission electron microscopy; (iii) morphometry allowing statistical analysis.
RESULTS: Submucosa of all LFN-exposed animals exhibit increased thickness with fibrous proliferation. Transmission electron microscopy showed massive collagen deposition. Immunostaining identified collagen IV as responsible for the increased thickness. Morphometry allowed the demonstration of a significant difference of thickness between control and exposed groups. Vascular alterations included: i) intima proliferation and thickening, rupture of the internal elastic lamina, thrombotic changes; ii) thickening of the media; iii) after 9 weeks of LFN-exposure, we found new formed vessel presenting tortuous and twisted. There is a significant difference of arterial wall thickness between control and exposed groups.
CONCLUSIONS: Deeper layers of gastric wall undergo alterations, including fibrosis of the submucosa caused by collagen IV deposition, an early marker of neoangiogenesis. Vascular alterations included thickening and thrombotic phenomena, but also images of newly formed vessels. This study suggests that, at least in the stomach, LFN-induced fibrosis could be linked with neoangiogenesis.
Headings: Gastric mucosa, physiopathology. Noise effects. Microscopy, electron. Rats.
CONTEXTO: Só um reduzido número de estudos avaliou as alterações digestivas causadas pelo ruído de baixa frequência (RBF) e, na sua maioria, focados nas alterações da mucosa.
OBJETIVO: Avaliar as lesões morfológicas induzidas na parede gástrica pelo RBF, para além da camada mucosa.
MÉTODOS: Ratos Wistar foram expostos a RBF, por períodos crescentes, desde 1 a 13 semanas. Um grupo controle foi mantido em silêncio. Fragmentos da parede gástrica foram estudados com recurso a: (i) microscopia ótica usando hematoxilina-eosina e imunomarcação para colágenos; (ii) microscopia eletrônica de transmissão; (iii) morfometria, permitindo a colheita de dados para análise estatística.
RESULTADOS: Observaram-se espessamento e proliferação de tecido fibroso na submucosa de todos os animais expostos aos RBF. A microscopia eletrônica de transmissão mostrou marcada deposição de colágeno. A imunomarcação identificou o colágeno IV como responsável pelo espessamento da submucosa. A morfometria permitiu demonstrar que a diferença de espessura da submucosa entre o grupo controle e os grupos de animais expostos era estatisticamente significativo. As alterações vasculares incluíram: (i) proliferação e espessamento da íntima, com rotura da lâmina elástica interna e fenômenos trombóticos; (ii) espessamento da média; (iii) após 9 semanas de exposição ao RBF, observaram-se vasos neoformados tortuosos e contorcidos. A espessura dos vasos dos animais expostos ao ruído é significativamente maior que a espessura no grupo controle.
CONCLUSÕES: As camadas profundas da parede gástrica sofrem alterações que incluem fibrose causada pela deposição de colágeno IV, marcador precoce de neoangiogênese. As alterações vasculares incluem espessamento e fenômenos trombóticos e vasos neoformados. O presente estudo sugere que, pelo menos no estômago, a fibrose associada ao RBF pode estar ligada à neoangiogênese.
Descritores: Mucosa gástrica, fisiopatologia. Efeitos do ruído. Microscopia eletrônica. Ratos.
In our modern urban societies, noise is a well recognised environmental hazard. For legal purposes, the sound description uses a unit called A-decibel, dB(A). As the human ear has a reduced sensitivity to sound in the frequencies under 500 Hz, a sound analyser is used, with a special filter that "mimics" the human ear evaluation of sound (A-weighting). Although useful to prevent auditory diseases, dB(A) underestimates low frequency noise (LFN) and dB(A) measurements are inappropriate to understand noise systemic effects. In order to evaluate these noise-induced systemic effects the "linear-weighting" must be used. Using "linear-weighting", expressed in linear decibel, dB(L), every sound frequency in the spectrum of 10-20 000 Hz has the same sound attenuation, a physical process similar to the one that occurs when sound reaches organs and tissues. As dB(A) measurements are used for legal evaluations, environmental LFN is usually higher than measured and frequently overlooked. The scientific community has long recognized the under evaluation of LFN(21). World Health Organization repeatedly addressed the public authorities and recommended the use of more accurate noise measurements(3).
Most of the published medical research on noise-induced diseases is related to hearing disorders. Nevertheless, non-auditory effects of noise have been reported for decades, including gastrointestinal (GI) clinical complaints and gastric lesions. Gastric lesions were found after chronic(1) and acute(5) noise aggression, both in humans(29) and animals(1, 29). A study performed among boiler-plant workers reported GI complaints, including heartburn, stomach cramps, nausea and diarrhea(13). After the implementation of a hearing protection program, these GI complaints increased, suggesting that the hearing protection, decreasing the audible exposure to noise, also decreased the avoidance of noise. This would increase the exposition to noise sources, which could be harmful through non-auditory pathways.
In the last three decades, most of these non-auditory effects of noise were related to the systemic effects of LFN. These included neurologic(31, 34, 41, 42, 48, 49), cardiac (7, 33), vascular(17, 35) and respiratory disorders(10, 16, 44, 45, 46). Lesions of the parotid gland(47) and periodontium of LFN-exposed rats were recently reported(38). Regardless of the organs and tissues studied, the LFN-induced morphologic alterations seem to occur with significant fibrosis(9, 17, 35, 47) and with major vascular lesions(17, 35, 47).
LFN-induced disorders include clinically identified digestive symptoms(6, 8). Using LFN-exposed Wistar rats, we disclosed massive cell death leading to diffuse gastric erosions(14, 19). Using the same experimental model, we proved that the duodenal mucosa also developed superficial erosions(15). Nevertheless, these upper GI lesions were restricted to the superficial mucosal layer. It was never reported any clinical or experimental study addressing the thickness of the gut wall, where important morphologic alterations could be expected.
The goal of the present study was to investigate the morphological injury of the gastric wall, beyond the epithelial layer, using light microscopy (LM), and transmission electron microscopy (TEM).
We used adult Wistar rats weights ranging from 210 g to 292 g, exposed to continuous LFN, during increasing periods:
Group 1 - 1 week (168 hours) - 10 rats
Group 2 - 3 weeks (504 hours) - 10 rats
Group 3 - 5 weeks (840 hours) - 10 rats
Group 4 - 9 weeks (1512 hours) - 10 rats
Group 5 - 13 weeks (2184 hours) - 10 rats
Each group had 5 males and 5 females. A control group (group C), of 15 rats (7 males and 8 females - weights from 209 g to 292 g), had the same experimental conditions, but was kept in silence. They were housed in groups of 2 or 3 rats per cage, in which they could move around. They had light cycles of 12 hours day/12 hours night. All animals were fed with standard rat food, had unrestrained access to water, and were treated according with the EU directive on Animal Protection for Experimental and Scientific Purposes (86/609/EEC).
Low frequency noise exposure - experimental protocol
The rat cages were placed in front of a noise generator, consisting of a sub-woofer Magnat xtc 1200, in an isolated compartment measuring 217 × 211 × 195cm. A computer Compaq (Pentium 133 MHz) reproduced a "white noise" previously recorded. The sound signal was amplified with a QSC amplifier and frequency-filtered, creating an acoustic environment rich in low frequency components. The noise exposure protocol was previously described(14). Noise level and the spectral analysis, similar in the entire compartment, were analysed by a digital real time analyser, near the position of the LFN-exposed rats. Levels were above 90 dB in the frequencies ranging from 50 to 500 Hz.
Rats were sacrificed with a lethal intraperitoneal injection of pentobarbital. After removing a small fragment for electron microscopy, the stomach was removed using a procedure similar to the one described by Nakamura et al.(43). Sections were stained with haematoxylin-eosin. Immunostaining for collagens I, IV and V was performed using the peroxidase technique.
Transmission electron microscopy
For TEM, the specimens were washed in phosphate buffer saline, fixed at room temperature in an aldehyde mixture made up of 4% formaldehyde, 1.25% glutaraldehyde and 10 nmol/L Ca Cl2 in 0.05 mol/L cacodylate buffer. The samples were dehydrated in ethanol and cut with a LKB microtome. The preparations were stained with uranyl acetate - lead citrate and examined in a JEOL 100C transmission electron microscope.
Morphometry was performed using the OPTIMAS software that allows an easy measurement of lengths and areas. In each rat, three artery images, stained with haematoxylin-eosin, were evaluated. In each artery we defined the lumen area, the wall area and the wall/lumen ratio (W/L ratio). Globally, we studied 45 arteries of control group rats and 30 arteries in each one of the 5 LFN-exposed groups. With the same software, thickness of gastric submucosa was measured in the proximal rat gastric wall (forestomach), where wall thickness is rather regular. Statistic evaluation was performed using the SPSS 13.0 program. For the W/L ratio we used the Scheffé "Post-hoc" test. For submucosal thickness evaluation we used the Welch and Brown-Forsythe tests and the Tamhane and Dunett tests.
We disclosed several morphologic changes on the gastric wall of LFN-exposed rats.
Observed with LM, submucosa of all LFN-exposed animals exhibit increased thickness, more striking in the rats exposed to noise for longer periods. This increased thickness was caused by fibrous tissue proliferation that frequently invaded the muscular layer. The TEM images revealed the collagen proliferation responsible for enlargement of the submucosa (Figure 1). Staining for collagens revealed that, in control rats, collagen I (Figure 2a) and collagen V (Figure 3a) were very abundant and collagen IV was hardly found and just in perivascular topography (Figure 4a). The submucosa of LFN-exposed rats revealed an almost complete disappearance of collagen I (Figure 2b) and collagen V (Figure 3b). In contrast, collagen IV was greatly increased (Figure 4b) and appeared to be responsible for the increase of gastric wall thickness. Morphometry confirmed the increasing width of the submucosa (Table 1), starting in the earlier exposure periods. The differences between the submucosal thickness of control group and each one of the noise-exposed groups were statistically significant (P<0.05). With progressive exposure the submucosa increased its thickness.
Gastric vessels and most remarkably submucosal arteries reveal significant alterations after a minimum of 3 weeks of LFN-exposure (groups 2 to 5). These alterations included: i) focal proliferation and thickening of the intima, with rupture of the internal elastic lamina, sometimes associated with thrombotic changes (Figure 5), ii) regular thickening of the media layer presenting eosinofilia and vacuolization (Figure 5) iii) in groups 4 and 5 (9 or 13 weeks of LFN-exposure), blood vessels on greater number, arteries and also veins were tortuous, convoluted and twisted (Figure 6), sometimes overlapping. These vascular changes were also present in other layers, namely in the adventitia. Morphometric evaluation confirmed the increasing W/L ratio, an indirect assessment of increasing arterial wall width exhibiting a progressive W/L ratio (Table 2). Group 1 had a W/L ratio similar to control group. Groups 3, 4 and 5 displayed a W/L ratio greater then control group and the Scheffé "Post-hoc" test demonstrated a statistically significant difference (P<0.005).
Reports had long linked non-auditory effects of noise with digestive complaints(13) and GI lesions(1, 29). Also, the spectra of the clinical disorders caused by LFN includes gastric and gut symptoms(8, 11). Using the Wistar rat model, we had previously proved that the proximal gut suffered with LFN-exposure developing diffuse gastric(14, 19) and duodenal(15) erosions caused by considerable epithelial cell lesion and death(14, 19). These epithelial cell lesions were similar to lesions previously found in other LFN-exposure tissues, namely the rat pleura(44, 46) and the bronchi(10, 16, 45). These lesions were also similar to ulcerative lesions caused by whole-body vibration (WBV), a vibratory phenomenon comparable to noise. Most of the clinical manifestations of the "WBV disease" are related with bone and joint trauma(4, 5) causing debilitating back pain(27, 28, 30) but digestive complaints are also frequent(39, 53). Lesions were found in a large group of WBV-exposed workers(55), using endoscopy and biopsies. Nakamura et al.(43), with an experimental model, produced WBV-induced gastric ulcers and proved that those ulcers were caused by direct mechanical effect of gastric vibration. Nevertheless, previous human and animal studies on LFN or WBV focused only the gastric mucosa and did not provide any understanding on the effects of vibration in deeper layers of the stomach.
Looking deeper into the gastric wall, the present study disclosed two main types of alterations of submucosa and other layers: vascular alterations and fibrosis. We found proliferation and thickening of the intima and the media, with rupture of the internal elastic lamina and thrombotic changes. These lesions were similar to those disclosed in other vessels, arteries, veins and lymphatics exposed to this kind of noise(17, 35, 47). Arterial lesions in the gastric submucosa were also similar to those reported in the "hand-arm vibration syndrome" that is a well known occupational disease related with the use of vibrating tools(37). Although joint (40, 56) and neural(22, 32, 51, 52) lesions are important, the syndrome seems to have a vascular pathogenesis. Clinically, it is characterised by recurrent vasospasm similar to Reynaud phenomenon(2, 12, 18, 36, 54). Biopsies of these patients revealed thickening, fibrosis and destruction of elastic fibres(57) analogous to the lesions found in the vessels of LFN-exposed rats in the present and previous studies(17, 35, 47). The resemblance of vascular lesions occurring in LFN-exposed and directly transmitted vibration is expectable since the physical phenomenon underlying directly transmitted vibration is the same of noise and sound. Only the auditory system allows the perception of air borne vibration as sound or noise. Reaching other organs, air borne vibration is similar to directly transmitted vibration. These mechanical forces could be responsible for the destruction of elastic fibres seen in the arteries of "hand-arm vibration syndrome" patients and could cause some of the lesions observed in LFN-exposed arteries, as the rupture of the internal elastic lamina.
The other important alteration disclosed was the massive proliferation of fibrous tissue. From previous reports in other organs, we already knew that LFN-exposure was related with major fibrosis(9, 17, 20, 33, 35, 47, 50). In our model, we disclose the presence of fibrous tissue enlarging the gastric submucosa of all exposed animals. Morphometric analysis proved that submucosal thickness of noise-exposed groups is significantly larger than submucosal thickness of control group.
The same mechanical forces that cause fragmentation of the elastic fibres of the arterial wall may be the cause of collagen production. Ingber(23, 24, 25, 26) has proved that mechanical forces applied to individual cells could change cell reactions to biochemical stimuli or even induce entirely different cellular responses. Mechanical forces resulting from tissue vibration may be the initial stimulus for collagen production. Although we could not clearly identify the triggering sign of the fibrotic process, the nature of the collagen production was enlightened. The use of specific collagen immunostaining identified collagen IV as the main contributor to this increased fibrous tissue. It is known that collagen IV appears early after an aggression, associated with neoangiogenesis, and later in the repairing process new formed vessels become visible and collagen I replaces collagen IV(58). In our model, the relationship between collagen IV and angiogenesis points towards a pathophysiologic linkage between these vascular lesions and the fibrosis. In the first weeks of LFN-exposure, vessels, most strikingly arteries, start showing thickening and thrombotic lesions. Simultaneously fibrotic changes start early: the thickness of the submucosa of 1 week exposed rats is significantly larger than the submucosa of control rats. Unlike other experimental models in which the aggression is time limited(58), in our model the LFN-exposure is continuous. Consequently, the fibrotic process is constantly induced and thickness increases with exposure. In the rats exposed for longer periods, group 4 (9 weeks) and group 5 (13 weeks) we observed simultaneously two features: as the persistent LFN-exposure is constantly stimulating the production of new collagen we found recently formed collagen IV, representative of the early stages of angiogenesis, but also an increased number of new vessels, frequently tortuous, convoluted and twisted, the end result of the neoangiogenesis pathway developing during 9 or 13 weeks. In the gastric wall exposed to LFN, fibrosis is due to collagen IV production and related with the neoangiogenesis process.
In the present study we proved that LFN-induced gastric lesions were not limited to the mucosa. Deeper layers, mainly the submucosa, also suffer important morphologic alterations. These included vascular lesions, namely thickening of the arterial walls with thrombotic phenomena. Fibrotic changes of the submucosa were caused by increased collagen IV production as part of a neoagiogenesis pathway resulting in new vessels formation after longer LFN-exposure periods. Although the presence of vascular lesions and fibrosis were long known features of LFN-exposed tissues, this study established a link between these two phenomena. At least in the gastric wall, the present data points to the collagen IV production occurring during a neoangiogenesis pathway as an important feature of the LFN-induced fibrosis. Further studies addressing hollow organs are needed, in order to evaluate the importance of neoangiogenesis in structures exposed to this kind of noise.
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Dr. Jorge Fonseca