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Keywords:

  • tendinopathy;
  • acute inflammation;
  • extracellular matrix;
  • collagen;
  • injury

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. LITERATURE CITED

Tendinopathy is a pathology found mainly in the rotator cuff, patellar, Achilles and flexor tendons. Tendinopathy is a significant impediment to performance in athletes and in workers in the labor market. Some studies have indicated that inflammation in adjacent tissues may affect the rotator cuff and Achilles tendon. In this study alterations were verified in the extracellular matrix (ECM) of the deep digital flexor tendon after two periods (12 and 24 hr) of induction inflammation in rat paw. Wistar rats were divided into three groups: those that received injection of 1% carrageenan; those that received 0.9% NaCl; and those that received no application. The tendon was divided into distal (d), proximal (p), and intermediate (i) regions. Biochemical analyses were performed and included non-collagenous proteins (NCP), glycosaminoglycans (GAGs), hydroxyproline (HoPro) and metalloproteinases 2 and 9. Tissue sections were stained with toluidine blue, hematoxylin-eosin, and Ponceau SS and observed under polarization microscopy. Remarkable results were detected that included the presence of MMP-9, degradation of NCP and GAG and the presence of cellular infiltrate closer to digits in d region. The different concentrations of HoPro, as well as alterations in the organization of the collagen fibers showed the collagenous matrix undergoing some alterations. The results indicated that the induced inflammation in rat paw exhibited characteristics similar to the typical acute inflammatory process observed in tendons. Anat Rec, 2013. © 2013 Wiley Periodicals, Inc.


INTRODUCTION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. LITERATURE CITED

The deep digital flexor tendon (DDFT) is classified as a “wrap around” tendon for being close to bone pulleys, which is subjected to different loads according to its extension. This tendon is usually divided into three regions, due to its biochemical and structural characteristics: distal region (d), subject to tension and some compression load; intermediate region (i), subject to compression load since it is in direct contact with the calcaneus bone; and proximal region (p), subject only to tension load (Benjamin et al., 2008; James et al., 2008). These regions are biochemically distinct, varying in the quantity and disposition of the components of the extracellular matrix (Covizi et al., 2001; Feitosa et al., 2006).

The prevalence of tendon and ligament injuries has increased in recent years. Athletes and workers are leaving the labor market because they are unable to perform their activities due to these disorders. Inflammation is perhaps the most common feature that affect tendons that may ultimately rupture (Clutterbuck et al., 2010).

When inflammation is diagnosed early, the tendon might restore its functionality after appropriate treatment. In general, tendinopathy is associated with repetitive and mechanical overloads. Although common, it is difficult to treat. This pathology is found mainly in the rotator cuff, patellar, Achilles, and flexors tendons (Tillander et al., 2001; Riley, 2008).

During the inflammatory process in the tendon there are variations in the contents of proteoglycans and collagens (Riley, 2008). Some enzymes, such as MMP-2 and 9, are involved in the remodeling process and degradation of extracellular matrix. These gelatinases participate in the degradation of denatured collagen, previously degraded by other enzymes (Karousou et al., 2008). Cellular infiltrates, composed mainly by inflammatory cells, are commonly noted among the collagen fibers (Sharma and Maffulli, 2006).

Carrageenan is a polysaccharide formed by repeated monomers that have groups of galactose sulfate, extracted from the cell wall of Rhodophyta algae such as Chondrus crispus (Campo et al., 2009). It has being widely used as an inductor of inflammation (Morris, 2003; Cicala et al., 2007, Vieira et al., 2012a,b). Inflammatory agents have been used to induce tendinopathies in experimental models used to study the efficacy of various potential therapies.

However, inflammation is not always present in the tendon, rather it may be in the adjacent tissues. These inflamed tissues may affect the metabolism of the extracellular matrix in the adjacent tendon. In a previous study, it was verified that acute inflammation can change the structure and biochemistry of the DDFT and Achilles tendon during the peak of inflammation in the rat pay within 4 hr after the administration of the inducing agent carrageenan (Vieira et al., 2012a,b). However, later effects are not established at this time. Therefore, in this study, acute inflammation was induced by carrageenan in the rat paw and the biochemical and structural alterations that occurred in the deep digital flexor tendon after 12 and 24 hr of inducing the inflammatory agent were evaluated.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. LITERATURE CITED

Experimental Groups

Wistar male rats 40 days of age, weighing 140–160 g, were kept with free access to water and food and on 12 hr light cycles. The rats were divided in three groups: (1) Animals that received subcutaneous injection of 1% carrageenan type IV (0.1 mL) dissolved in saline, according to Winter et al. (1962). (2) Animals that received vehicle (0.1 mL 0.9% NaCl). (3) Animals that received no application. Carrageenan was injected into the footpad of the right paw of the rats and only the right tendon was removed for analysis. For this, the animals were anesthetized using isoflurane (Forane). After 12 and 24 hr, the animals in the respective groups were killed by an overdose of the same anesthetic agent and tendons removed for morphological and biochemical analyses (n = 5 tendons analyzed). Animal care is in accordance with the ethical principles of animal experimentation adopted by the Brazilian College of Animal Experimentation (COBEA) and the study was approved by the Ethics Committee on Animal Experiments of State University of Campinas, SP, Brazil.

Extraction of Extracellular Matrix

After the DDFT was removed from the right hindpaw of each animal, it was divided in three regions (d: distal, i: intermediate and p: proximal). Each region was analyzed individually. The tendons were thoroughly washed with PBS solution containing 0.15 M NaCl, 5 mM sodium phosphate buffer pH 7.4 with 50 mM EDTA. The regions were cut into small pieces and immersed in 4 M guanidine hydrochloride containing 0.05 M EDTA and 1 mM PMSF in 0.05 M acetate buffer pH 5.8, according to the method of Heinergard and Sommarin (1987). The extraction lasted for 24 hr at 4°C followed by centrifugation at 27,000g for 50 min. The supernatant was used for biochemical analysis.

Agarose Gel Electrophoresis

The fragments of the tendons were dehydrated and sulfated glycosaminoglycans were released from proteoglycans by digestion with a papain solution (40 mg/g of dry tissue) containing 100 mM sodium phosphate buffer, pH 6.5, 40 mM EDTA and 80 mM β-mercaptoethanol (Harab and Morão, 1989). The sulfated glycosaminoglycans (GAGs) were precipitated in ethanol and separated by agarose gel electrophoresis (0.6%) in 0.05 M propylenediamine (PDA) according to Dietrich and Dietrich (1976).

Quantification of Hydroxyproline

The fragments of the three regions of the DDFT were immersed in acetone for 48 hr and then in chloroform: ethanol (2:1) for 48 hr. Thereafter, the fragments were hydrolyzed with 6 N HCl (1 mL/10 mg tissue) for 16 hr at 110°C. The hydrolysate was neutralized with 6 N NaOH and treated with a 1.41% solution of chloramine-T and 15% p-dimethylaminobenzaldehyde as described by Stegemann and Stalder (1967). The solution was cooled at room temperature, and the absorbance was read at 550 nm using a Diode Array spectrophotometer (Model 8452A, Hewlett Packard).

Quantification of Proteins and Sulfated Glycosaminoglycans

The guanidinium chloride extracts collected from the experimental groups were used to quantify the relative amounts of protein according to the method of Bradford (1976). Bovine serum albumin was employed as a standard. The samples digested by papain solution were used to quantify the GAGs of the experimental groups. The quantification was determined using the dimethylmethylene blue (DMMB) method (Farndale et al., 1986) using chondroitin sulfate as the standard. The absorbance was measured at 595 nm for proteins and at 540 nm for glycosaminoglycans using an Asys Expert Plus Microplate Reader.

Zymography for MMP-2 and MMP-9

The tendons were treated according to the method described by Marqueti et al. (2006). For protein extraction, the fragments from each region of the DDFT were immersed in a solution of 50 mM Tris-HCl pH 7.4, 0.2 M NaCl, 10 mM CaCl2, and 0.1% Triton with 1% of a protease inhibitor cocktail (Sigma) at 4°C for 2 hr. After the first extraction, the samples were incubated and added to 1/3 the volume of the solution described above, at 60°C for 5 min. Then, 20 μg of the protein extract was loaded on the gel. The protein samples were electrophoresed at 4°C on a 10% polyacrylamide gel containing 0.1% gelatin, and after completion of electrophoresis, the gel was washed with 2.5% Triton X-100 and incubated for 21 hr in a solution of 50 mM Tris-HCl (pH 7.4), 0.1 M NaCl and 0.03% sodium azide at 37°C. The gel was stained with Coomassie brilliant blue R-250 (Sigma) for 1 hr. After staining, the gels were washed with a solution containing 50% methanol and 10% acetic acid to observe negative bands of proteins corresponding to enzymatic activity. As a positive control, 20 mM EDTA was used in the incubation buffer. EDTA inhibits the activity of gelatinase and confirms the identification of MMPs in the gels. The bands in the negative image were quantified by densitometry using Scion Image software Alpha 4.0.3.2 (Scion Corporation).

Light Microscopy Analysis

The tendons were fixed in a solution containing 4% formaldehyde in Millonig buffer (0.13 M sodium phosphate, NaOH 0.1 M, pH 7.4) for 24 hr at room temperature. Afterward, the tendons were washed for 6 hr in tap water, dehydrated in ethanol, diaphonized with xylene and embedded in paraffin (Histosec, Merck), according to Neto et al. (2003). Longitudinal serial sections were cut at 7 µm for microscopic analysis. Some sections were stained with hematoxylin-eosin (HE) and others with toluidine blue (TB) (0.025%) in McIlvaine buffer (0.03 M citric acid, 0.04 M sodium phosphate dibasic, pH 4.0). Sections stained with HE and TB were observed on a light microscope (Olympus BX 60).

Polarizing Microscopy

To visualize the arrangement of the collagen fiber bundles, the sections were stained with 0.025% Ponceau SS and 2% acetic acid for 1.5 min and the Linear Dichroism (LD) was observed on a polarization microscope (Vidal and Mello, 2005). When the collagen bundles are highly oriented, some alterations in this organization may be detected by observing the difference in the light absorbance when the long axis of the tendon is parallel or perpendicular to the polarized light plane (Linear Dichroism). As a result differences in the images between parallel and perpendicular plans in relation to the polarized light is detected (Aro et al., 2008; Vieira et al., 2012a).

Statistical Analysis

Data were presented as mean ± SD of results obtained from five animals per group. To compare the data, statistical analysis was performed using one-way analysis of variance (ANOVA) with the Tukey post hoc test. A value of P < 0.05 was considered statistically significant, and the statistical program GraphPad Prism®, version 3.0 was used for all analyses.

RESULTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. LITERATURE CITED

The three regions (distal, intermediate and proximal) of the DDFT were analyzed individually and characterized by biochemical and morphological analysis.

A lower concentration of non-collagenous proteins in the animals from the carrageenan group, at 12 hr, was observed in the d region when compared to the control and the vehicle groups as seen in Fig. 1A. However at 24 hr the i region in the carrageenan group exhibited lower relative amounts of non-collagenous proteins while the p region higher relative amounts when compared to the control and vehicle groups. This was significantly different compared with the carrageenan group at 12 hr. In the protein profile through SDS-PAGE, no differences were detected between the experimental groups (data not shown).

image

Figure 1. Quantification of components extracellular matrix of DDFT: (A) Noncollagenous proteins: A smaller amount of noncollagenous protein was noted in animals that received the application of carrageenan in d and i region. In p region was observed a high concentration of noncollagenous protein; (B) Concentration of sulfated glycosaminoglycans: Observe higher amount of glycosaminoglycans in d region in Car group, and lower concentration in the Veh group; (C) Concentration of hydroxyproline: After 12 hr, a high concentration was detected in the i and p region in the Car group compared to control. After 24 hr, a high concentration was observed in d region when compared to vehicle and control. A high concentration of hydroxyproline was noted in d and p region after 12 hr in the Veh group.* Significant difference in relation to control, # significant difference in relation to the vehicle.

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The total amount of sulfated GAG in the d region after 12 hr was greater than in the vehicle group. In the i region, a lower concentration was detected in vehicle groups compared to control (Fig. 1B). The levels of individual GAGs were analyzed by agarose gel electrophoresis. It was observed that the dermatan sulfate band was less prominent in the i region of the carrageenan group after 12 hr (Fig. 2A). Meanwhile, at 24 hr the same band appeared as in normal conditions (Fig. 2B), when verified according to band densitometry (Table 1).

image

Figure 2. Agarose gel electrophoresis in A (12 hr) and B (24 hr): HS (heparan sulfate), DS (dermatan sulfate), and CS (chondroitin sulfate) standards are in the left. In (A), observe in the i region the DS band was less pronounced in carrageenan group (Car) at the 12 hr. Therefore, in 24 hr (3B), the same band return the normal conditions. There were not significantly differences in relation the other groups.

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Table 1. Densitometry of dermatan sulfate bands corresponding to deep digital flexor tendon of the experimental groups
Groupsd regioni regionp region
  1. a

    P < 0.05 relative to control and vehicle.

Control 12h85134 ± 316601 ± 130,6688 ± 174,6
Vehicle 12h761,3 ± 243,10724 ± 229670 ± 12,49
Carrageenan 12h889,4 ± 19535498 ± 52,57a554,6 ± 66,04
Control 24h779 ± 125,70716,5 ± 137,24890 ± 26,86
Vehicle 24h816,7 ±17,01706 ± 109,46782 ±112,40
Carrageenan 24h983 ± 190,89713 ± 93,19930 ± 128,60

A higher concentration of hydroxyproline was detected in the i and p regions of the carrageenan group when compared to the control after 12 hr (Fig. 1C). It was possible to observe that the vehicle group presented a greater concentration of hydroxyproline in the d and p regions when compared to the control, although no differences were observed relative to the carrageenan group. However, at 24 hr, the d region was greater when compared to the control and vehicle groups. In general, at 24 hr lower concentrations of hydroxyproline were observed in vehicle and carrageenan groups in all regions when compared to the experimental groups at 12 hr.

No differences were observed in the levels of the three isoforms of MMP-2 in each region of DDFT in the different experimental groups at 12 and 24 hr (Table 2). However, it is noteworthy that the latent and active isoforms of the MMP-9 were present only in the d region of the animals treated with carrageenan after 12 hr (Fig. 3).

Table 2. Densitometry of MMP-2 and 9 of the DDFT regions
 d regioni regionp region
ParametersCVehCarCtrVehCarCtrVehCar
  1. a

    P<0.05 relative to control and vehicle groups.

12h         
MMP-2 active5839,33 ± 121612134 ± 1375,75225,67 ± 15421145,33 ± 2431970 ± 687,5775,33 ± 2308085 ± 2673,27080,67 ± 13514914,3 ± 1001,5
MMP-2 inter.31784,3 ± 1022037312 ± 3567,247769 ± 544623933,3 ± 9100,140800,6 ± 100131067,3 ± 2540,729794 ± 6426,933267 ± 794132583,6 ± 5932,7
MMP-2 latent26402,2 ± 554927143,6 ± 722423352 ± 3574,319566,6 ± 2910,322363 ± 4308,818941,3 ± 2128,317526,6 ± 708322521,6 ± 589512826,6 ± 4903
MMP-9 active4042 ± 1526,9a
MMP-9 latent4697,33 ± 965a
24h         
MMP-2 active5576,67 ± 23228567,33 ± 18192799,6 ± 952231,3 ± 27,1521,67 ± 191566 ± 141,72112,3 ± 8423573,6 ± 12002524,3 ± 1002,5
MMP-2 inter.68820 ± 1400083203,3 ± 140629380 ± 63814243,3 ± 15698703,3 ± 15693604,3 ± 66015712 ± 169819719,3 ± 319619628,6 ± 5161
MMP-2 latent25701 ± 800023633,6 ± 72963034 ± 800435,6 ± 82688 ± 83,6480 ± 119,22453,6 ± 704,33112 ± 4792825,6 ± 956
image

Figure 3. Zymography of MMP-2 and MMP-9 in extract of rat tendons. Observe there is active (83 kDa) and latent (92 kDa) isoform of MMP-9 in 12 hr after application of carrageenan in d region. These bands was not detected in 24 hr. It was not observed significant differences the activity of MMP-2 among the groups.

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Sections of tendons stained with TB showed a cellular infiltrate in a region close to the digits in the d region of the carrageenan group after 12 hr (Fig. 4H). This was not observed in the control and vehicle groups and in all groups at 24 hr. The presence of cellular infiltrate on the epitenon was evident in the tissue in the three regions of the DDFT, easily observed in sections stained with HE in carrageenan group after 24 hr (Fig. 4E).

image

Figure 4. Histological appearance of deep digital flexor tendon stained with hematoxylin-eosin (A–E) and toluidine blue (F–H): (A and F) control group; (B and D)12 and 24 hr vehicle group; (C and E) 12 and 24 hr carrageenan group, respectively; (F and G) 12 hr vehicle and control group; (H) carrageenan group. Note the thicker epitenon in E (*), when compared to other experimental groups. Bar: 50 μm. In TB, we noted after 12 hr, the cellular infiltrate ([RIGHTWARDS ARROW]) between collagen fibers in H. Bar: 40 μm.

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The analysis of the sections stained with Ponceau SS and observed by polarizing light microscopy showed a lower linear dichroism in the p region in the vehicle and carrageenan groups when compared to the control at 12 and 24 hr (Fig. 5). These results indicated less organization in the collagen bundles. In the d region, there did not appear to be a difference between the experimental group and the controls at 12 and 24 hr (data not shown).

image

Figure 5. Longitudinal sections of the p region stained with PSS and observed by polarizing microscopy. In A, B, and C are parallel cuts in D, E, and F are the cuts perpendicular to the plane of polarization. A and D: control group; B and E: vehicle group; C and F: carrageenan group. The same profile was detected in vehicle and carrageenan groups in 12 and 24 hr in relation the organization of collagen fibers. In relation to the control group, the other groups present a smaller linear dichroism. Bars: 20 μm.

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DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. LITERATURE CITED

In this study, the possible alterations in the ECM of the DDFT, after inducing the inflammation in rat paw by carrageenan were investigated. One of the most common protocols for inducing inflammation is by the administration of carrageenan. This approach is often utilized in experiments that aim to test new drugs against inflammation (Morris, 2003; Cicala et al., 2007, Campo et al., 2009, Vieira et al., 2012a,b).

It was apparent that after 12 and 24 hr after induction of inflammation in the rat paw that there were differences in the tendon response. The concentration of hydroxyproline was higher at 12 hr in the i and p regions of the carrageenan group when compared to the control. However, at 24 hr there were decreases in these regions in the carrageenan group relative to the control and vehicle groups. These results suggest that the inductions of inflammation in the rat paw directly affects the amino acids that are predominant in the collagen molecule. In the d region, after 24 hr, a high concentration of hydroxyproline was observed in the carrageenan group compared to vehicle and control groups. This suggests that during this period, the d region is still in the reorganization process after the induction of inflammation. Otherwise, the vehicle and carrageenan group (i and p region) approached the concentration of hydroxyproline noted in control group after 24 hr.

The vehicle and carrageenan groups had a greater quantity of hydroxyproline in d and p region; i and p region, respectively relative to the control group at 12 hr, thereby indicating that the application with saline solution on the rat paw may alter the concentration of this extracellular matrix component. It is known that ECM fibroblasts release different growth factors and can synthesize collagen in response to different challenges (Midwood et al., 2004; Wang, 2006; Aro et al., 2008).

The latent and active isoforms of the MMP-9, an enzyme characteristic of the inflammatory processes (Chakraborti et al., 2003; Clutterbuck et al., 2010) were detected in the d region after 12 hr of the carrageenan application. The MMP-9 may have been responsible for the decrease in the quantity of hydroxyproline in the d region at 12 hr, given that this enzyme degrades native and denatured collagen, proteoglycans and glycoproteins (Gill and Parks, 2008; Clutterbuck et al., 2010). In a previous study, only the active isoform of the MMP-9 was observed at 4 hr after the induction of inflammation (Vieira et al., 2012a). It should be noted that in the present study at 24 hr, the MMP-9 disappears. These data indicated that the profile of this enzyme changes during the first day after induction of inflammation in the rat paw.

The lower quantity of non-collagenous proteins in the d region and the smaller presence of dermatan sulfate band in the i region at 12 hr in the carrageenan group may be due to an increase of degradation rates, a typical feature of tendinopathy (Riley, 2008). In a companion study, lower rates of proteins in all regions that received inflammation in adjacent tissue were detected at 4 hr after application of carrageenan (Vieira et al., 2012a).

In the i region of tendons from the carrageenan group, the dermatan sulfate band showed the same presence as the control group at 24 hr. This suggested that the tissue is returning to normal, however, in the p region a higher concentration of the protein was noted. This would suggest that this region was not completely reorganized during this time period.

Analyses of sections stained with PSS and observed by polarization microscopy showed that in the d region the collagen fiber bundles appeared to have the same organization in all control groups at the 12 and 24 hr period. However, differences were noted at 4 hr, in the same region in a prior study we noted a greater aggregation of collagen fibers arranged in a highly organized manner (Vieira et al., 2012a). These combined observations would suggest that after 12 and 24 hr there were organization of the collagen fiber bundles has returned to their basal, normal condition.

However, there were some differences noted in the p region that included less organized pattern in the vehicle and carrageenan groups. As the p region is mainly under tension forces, and the collagen bundles are highly oriented, some alterations in this organization may be detected by observing the difference in the light absorbance when the long axis of the tendon is parallel or perpendicular to the polarized light plane (Linear Dichroism). The collagen bundles are disposed parallel in normal condition, but under pathological processes such as inflammation, lesion, stress and stretching, the ECM components undergo structural reorganization of the tissue (Aro et al., 2008; De Aro et al., 2012; Vieira et al., 2012a).

The d region in carrageenan group had a small cellular infiltrate at 12 hr. The epitenon was thicker at 24 hr in this group relative to the control and vehicle groups. The cellular infiltrate in injured tissue is a typical inflammatory process and has the function of establishing the homeostasis of the tissue (Vaday and Lider, 2000; Adair-Kirk and Senior, 2008). It is known that the MMP-9 can be secreted by inflammatory cells (Clutterbuck et al., 2010). Thus, in the peak of inflammation (which appear to be about 4 hr after application of carrageenan), it was possible to observe the thicker epitenon in all the regions of DDFT that also contained larger amount of cells (Vieira et al., 2012a). In other companion study, cellular infiltrates in the Achilles tendon was not observed (Vieira et al., 2012b). Different tendons, functionally distinct, have different rates of metabolism (Birch et al., 2008) and may also respond to the inflammatory processes in different manners. Each region of the DDFT is distinct in relation to the results found in the composition of their ECM. Probably, in the region closer (d) to the site of inflammation, more pronounced changes may be observed. The inflammation of tissues surrounding tendons has been studied. For example, Tillander et al., (2001) induced bursitis, and showed a disorganization of the collagen molecules, macrophages infiltration. Additionally, in some animals they noted fibrocartilaginous tissue and bony metaplasia within the supraspinatus tendon. In the Achilles tendon, alteration in the MMP-2 activity have been observed and showed a tendency in degradation of ECM composition after induction of inflammation (Vieira et al., 2012b). Cellular infiltrates in the epitenon, alterations in collagen fiber organization, degradation of non-collagenous proteins and glycosaminoglycans, presence of MMP-9, alterations in relation the hydroxyproline in tissue, were also observed after 4 hr of application of carrageenan on rat paw (Vieira et al., 2012a).

Some of the results presented were similar to established characteristics of acute inflammatory processes (Marsolais et al., 2001; Riley, 2008). This would include the presence of MMP-9, cellular infiltrates and degradations of some ECM elements. This study, combined with results from prior studies, describes so of the alterations that occur in the extracellular matrix elements over times up to 24 hr after induction of inflammation. Regional differences in the tendon were observed and described. As observed in this study, some of the inflammatory indicators appeared to be at least partially reversed by 24 hr after carrageenan administration to the rat paw.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. LITERATURE CITED

The authors thank Francisco A. Mallattesta for his technical assistance. C.P. Vieira was the recipient of Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES - Brazil) fellowship.

LITERATURE CITED

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. LITERATURE CITED