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1.3 Microencapsulación de aceites

1.3.4 Secado por aspersión como un proceso para microencapsulación de

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The distribution o f matrix metalloproteinases 1, 2, 3, and 9

during corneal wound healing

Summary

Introduction

Methods

Results

Discussion

Summary

Purpose: To compare the distribution o f the matrix metalloproteinases (MMPs) -1 ,-2 , -3 and -9 after superficial keratectomy or lamellar keratectomy.

M ethods: Twelve rabbits were studied in each group. Wounds were 6 mm in diameter and made with a vacuum trephine. Superficial keratectomy wounds were 180 pm deep while lamellar keratectomy wounds were 240 pm deep but the intact comeal epithelium and anterior stroma (approximately 120 pm) were replaced. Animals were sacrificed at 4, 18, and 24 hours and at 3, 7 and 14 days. Labelled antibodies to MMP-1, -2, -3 and -9, were identified in frozen sections by indirect immunofluorescence.

R esults: MMPs-1 and -9 appeared early in the basal layer o f the migrating epithelium o f superficial keratectomy wounds with a peak expression at 3 to 7 days. MMP-2 was

detected in the anterior stroma and the basal epithelium. M M Ps-land -9 were not detected in Lamellar Keratectomy wounds, although MMP-2 was detected in the intrastromal wound from 4 hours with a peak at 3 days. MMP-3 was only weakly expressed at all time points.

Discussion: MMPs-1 and -9 are not expressed when stroma and epithelium are replaced following lamellar keratectomy. However MMP-2 is expressed at an intrastromal wound interface. MMPs-1 and -9 appear to be associated with re-epithelialisation after comeal wounding whereas MMP-2 is associated with the breakdown o f damaged extracellular matrix in the comeal stroma.

Introduction

Excimer laser photorefractive keratectomy (PRK) and laser in situ keratomileusis (LASIK) modify the radius o f curvature o f the anterior comeal surface to correct refractive error. Healing after PRK involves re-epithelialisation as well as stromal remodelling, whereas after LASIK the intact epithelium is replaced and there is correspondingly less remodelling at the wound interface than after PRK. Clinically, for a comparable refractive correction, there is less scar formation and haze following LASIK than PRK. (Jain S et al., 1995) (Hersh PS et al., 1998) Stromal scarring can lead to a reduction in visual acuity and regression o f the induced change in refraction. (Tuft SJ et al., 1989) (Tuft SJ et al., 1993) An understanding o f the mechanism o f this scarring process could lead to the development o f more accurate treatments.

MMPs are a group o f zinc dependent enzymes that degrade ECM and basement membrane components and are in dynamic equilibrium with their specific tissue inhibitors. They are involved in normal development and in wound healing as well as in disease such as Rheumatoid arthritis, cancer and metastasis. (Matrisian LM, 1990) Abnormal expression has been associated with persistant epithelial defects, and comeal ulceration. (Birkedal- Hansen H et al., 1993) In healing skin wounds MMP-1 is essential for the migration o f kératinocytes over a type I collagen matrix, (Pilcher BK et al., 1997) but little is known about its expression in the com ea after anterior keratectomy. Using zymography, Girard et al (1993) found an increase in collagenase (MMP-1) and stromelysin (MMP-3) synthesis for 4 weeks after a penetrating keratectomy. (Girard MT et al., 1993) Certain gelatinolytic MMPs have been found to be upregulated in the comea after PRK. Azar et al (1996 and

1998) found that MMPs-2 and -9 were upregulated in the epithelium and anterior stroma o f the rat com ea during wound closure after PRK. (Azar DT et al., 1996) (Ye HQ & Azar DT, 1998) The same group has used zymography to show that in the rabbit comea MMP- 9 is upregulated in the epithelium after PRK and not after LASIK, and they suggested that MMP-9 may have a role in the pathogenesis o f scarring after PRK. (Azar DT et al., 1998) Matrilysin (MMP-7) and Stromelysin (MMP-3) were recently immunolocalised in the wounded rat comea after PRK; MMP-7 was expressed in the leading edge o f the epithelium and MMP-3 restricted to the deeper stroma. (Lu PCS et al., 1999) It has previously been suggested that MMP-2 is important in long term tissue remodelling, and

that MMP-9 is involved in the control o f epithelial basement membrane resynthesis. (Matsubara M et al., 1991a) Such differences in the expression o f MMPs could indicate potential targets for specific inhibitors o f MMPs that could be used to achieve comeal refi*active surgery without scarring. We have therefore used immunohistochemistry to compare the distribution o f MMPs -1 ,-2 , -3 and -9 after superficial keratectomy or lamellar keratectomy to determine differences in expression and distribution during re- epithelialisation and the early phase o f stromal remodelling.

Methods

All animals received adequate care and humane treatment as stipulated by the ARVO statement for the use o f animals in Ophthalmic and Vision research. Twenty-four

pigmented Dutch belted rabbits weighing IKg to 1.5 Kg were anaesthetised before surgery see 2.2.1.

Surgical Procedure

Rabbits were divided into two groups; group I (n = 12) received SK see 2.2.1. and group II (n = 12) received LK see 2.2.3. After surgery all rabbits received topical G

Chloramphenicol 0.5% 4 times a day in their operated eyes until fully re-epithelialised (approximately 5 days). Animals were examined with the slit lamp at 18 hours, 24 hours, then daily until re-epithelialised, and then on days 7 and 14. Comeas were harvested at 4 hours, 18 hours, 24 hours, 3 days, 7 days and 14 days after surgery. Early time points were chosen in order to look at the distribution o f MMPs during the phase o f re-epithelialisation in the SK wounds compared with the non re-epithelialising LK wounds, and follow similar time points to previous published studies. (Azar DT et al., 1996) (Ye HQ & Azar DT, 1998) This study was not designed to look at differences in scar formation between the 2 surgical models and so later time points were not used.

Histology and immunohistochemistry

Corneoscleral buttons were processed for immunohistochemistry as in section 2.3.8. All the antibodies used, bind to pro and active enzyme forms o f the MMPs, as well as to active enzyme bound to the TIMP. However pro-MMP-1 cannot bind to the matrix whereas active MMP-1 can. Therefore MMP-1 staining indicated active or TIMP bound MMP-1. (Allan JA et al., 1991)The stromelysin (MMP-3) and Gelatinase (MMP-2 and -9) pro and active forms both bind to the matrix, therefore immunostaining does not

Results

Clinical examination

All the Superficial Keratectomy wounds had re-epithelialised by day 5. There were no cases o f secondary epithelial breakdown or infection. At day 14 comeal haze was less after lamellar keratectomy than after superficial keratectomy. The slit lamp appearance at 1 month afl;er wounding is shown in Figures 4:01 and 4:02.

Histological examination

From day 7 there was epithelial hyperplasia across all superficial keratectomy wounds. Hyperplasia was not present in the lamellar keratectomy specimens.

MMP expression

Superficial keratectomy

MMPs-1 and -9 were expressed in the basal epithelial area fi*om 18 hours. This was seen as a continuous fluorescent band o f MMP-1 staining under the migrating epithelial edge and MMP-9 staining slightly behind the leading edge. The staining was brightest at day 3, but between days 7 and 14 the staining had reduced and was no longer continuous in this area. Bright fluorescent spots appeared in areas where the epithelium was irregular and epithelial stromal attachment was less secure (Figure 4:03a and b and Figure 4:06a and b). The distribution o f MMP-2 expression was similar to that o f MMPs-1 and -9 but localised more to the anterior stroma than the basal epithelium (Figure 4:04a and b).

There was little expression o f MMP-3 at any time point (Figures 4:05a and b). Table 4:01 summarises the findings. (Table 4:01)

Lamellar Keratectomy

MMPs-1, -3 and -9 were not detected in the epithelium or anterior stroma o f the central wound (except at the very edge o f the wound where the epithelial surface was breached)

(Figures 4:03, 4:05 and 4:06). However MMP-2 was detected at the intrastromal wound interface fi-om 4 hours, and appeared to stain most brightly at day 3 (Figure 4:04).

24 hours

Table 4:01: Table o f Results -Corneal Matrix Metalloproteinase expression over time

Matrix Metalloproteinase Wound 4 hours 18 hours 24 hours 3 days 7 days 14 days

SK - + + + + -/ + LK - - - - SK - + 4 - 4 - 4 - - / + LK + + + + + -/ + SK - - - -/ + -/ + LK - - - - SK - + + + -/ + -/ + LK - - - -

[S K = superficial keratectomy, L K = lamellar keratectomy, - = no stain greater than control negative, -/+ = fa in t fluorescent staining, + = positive staining,

Controls

There was no staining for MMP-1, -2, -3 or -9 in the unwounded rabbit comea controls

(Figure 4:07)). Negative wounded controls (Figure 4:08) and positive and negative fibroblast controls (Figure 4:09) are shown.

The distribution o f staining was seen most clearly at the 18 hour time point. Larger scale images have been included for easier recognition o f the difference between the 2 wounded groups. (See Figures 4:10 to 4:17)

Normal cornea

MMP-2

MMP-3

MMP-9

Figure 4:07: Unwounded rabbit corneas stained with fluorescent labelled antibodies to

MMP-2, -3 and -9, (x 200)

Superficial

Negatives

Lamellar

Keratectomy

4 hours

18 hours

24 hours

Figure 4:08a: Negative controls o f superficial and lamellar keratectomy wounds at 4, 18 and 24 hours. (Arrows indicate site o f wound) (x200)

Superficial

Keratectomy

Negatives

Lamellar

Keratectomy

3 days

7 days

14 days

Figure 4:08b: Negative controls o f superficial and lamellar keratectomy wounds at 3y 7 and 14 days, (Arrows indicate site o f wound) (x200)

Negative ra b b it com eal fibroblast control

MMP-1 positive rab b it corneal fibroblast control

M MP-2 positive rab b it com eal fibroblast control

M M P-3 positive rab b it com eal fibroblast control

y

M M P-9 positive rab b it com eal fibroblast control

Figure 4:10 18 hours after superficial keratectomy MMP-1 (x 200). Showing bright fluorescent staining under the advancing edge o f the basal epithelium (Arrow).

Figure 4:1118 hours after lamellar keratectomy MMP-1 (x 200). Showing no staining o f the midstromal wound interface (Arrow).

Figure 4:12 18 hours after superficial keratectomy MMP-2 (x 200). Showing bright fluorescent staining o f the basal epithelium behind the advancing edge and in the anterior stroma (Arrow).

Figure 4:13 18 hours after lamellar keratectomy MMP-2 (x 200). Showing bright fluorescent staining at the mid stromal wound interface (Arrow).

Figure 4:14 18 hours after superficial keratectomy MMP-3 (x 200). Showing no stain at the anterior stromal wound site (Arrow).

Figure 4:15 18 hours after lamellar keratectomy MMP-3 (x 200). Showing no stain at the mid stromal wound interface (Arrow).

Figure 4:16 18 hours after superficial keratectomy MMP-9 (x 200). Showing bright fluorescent staining slightly behind the leading edge o f the migrating epithelium (Arrow).

9

Figure 4:1718 hours after lamellar keratectomy MMP-9 (x 200). Showing no stain at the mid stromal wound interface (Arrow).

Discussion

Our results show that in the rabbit there is a difference in the healing process between superficial and lamellar keratectomy wounds. Re-epithelialisation in the superficial

keratectomy wounds was associated with scar deposition whereas after lamellar keratectomy there is reduced scar formation. Increased MMP-1 and -9 expression during epithelial

migration after superficial keratectomy correlates with the proposed roles for MMP-1 in epithelial cell migration on collagen matrices and the importance o f MMP-9 for basement membrane assembly. MMP-2 expression in the stroma beneath the migrating epithelium could be important for remodelling o f the damaged ECM. In this study MMP-3 was not found in the deep stromal layers, but we did find expression in the anterior stroma at 3 to 7 days after superficial keratectomy. Previously MMP-3 has been found in the deep stromal layer during the first 3 days after excimer laser in a rat model and subsequently in the area o f remodelling stroma. (Lu PCS et al., 1999) It can be postulated that the differences fi*om our study could be due to species variation (rat versus rabbit model) and to the use o f the excimer laser (rather than a surgical technique) to create the wound. Azar et al, (1998) compared 2 different wound models using zymography to detect MMP-2 and -9 after PRK and LASIK and found

significantly higher MMP-2 and -9 in the epithelium after PRK. These findings are in agreement with our immunolocalisation studies. However they also found no difference in stromal MMP-2 expression between unwounded, PRK, LASIK and comeal flap treated corneas, (Azar DT et al., 1998) whereas we found increased MMP-2 at the intrastromal wound interface. This disagreement may be due to the differences in techniques used in the studies, both to create the wound and also in MMP detection. The higher incidence o f comeal haze after PRK compared with LASIK may be associated with the upregulation o f MMPs. (Azar DT et al., 1998) It has been postulated that the greater production o f certain MMPs, particularly MMP-9, after PRK compared with LASIK might explain the differences in scar formation.

After PRK although the amount o f tissue removed is very precise the subsequent wound healing response leads to epithelial hyperplasia and comeal scarring which results in

unpredictability o f the refractive outcome and haze. (Fantes FE et al., 1990) (Lohmann C et

a l, 1991) (Gartry DS et al., 1992a) (Gartry DS et al., 1992b) A number o f studies have shown that after surface injury to the cornea, either surgically or laser induced, new connective tissue is deposited beneath the epithelium in the anterior stroma. (Tuft SJ et al.,

1989) (Rawe IM et ah, 1992b) (Tuft SJ et al., 1993) This new ECM, is difierent to that present in the unwounded cornea. (Corbett MC et al., 1996) (Corbett MC & Marshall J,

1996) The resulting haze reduces with time as the scar tissue is re-modelled. (Tuft SJ et al.,

1987) (Tuft SJ et a l, 1989) (Rawe IM et a l, 1992b) (Tuft SJ et a l, 1993)

Many factors, in addition to MMPs, are important in the process o f wound healing after PRK and LASIK. In LASIK the central epithelium and anterior stromal layers remain intact, the intrastromal wound site is also protected from inflammatory cells and cytokines (e.g. EGF) contained in the tear film, and the structural integrity o f the epithelial BM layer is preserved over the optical zone. It is thought that there is less cytokine release from the epithelium than after PRK and a reduced stimulus for stromal keratocytes to undergo fibroblastic change. As there is normally less scarring and regression, LASIK has become the treatment o f choice for higher degrees o f myopia. (Khoury JM et a l, 1995) (Vale S et a l, 1995) (Hersh PS et a l,

1998) The maintenance o f the intact epithelium and basement membrane in LASIK appears to be associated with reduced scar formation. (Park CK & Kim JH, 1999) The cytokines that participate in the interaction between the comeal epithelium and stroma have been

investigated. Paracrine growth factors (e.g. HGF and KGF) produced by fibroblasts modify epithelial cells in the wounded cornea. (Wilson SE et a l, 1994) (Li Q et a l, 1996) (Wilson SE

et a l, 1996b) It has been suggested that IL -l a released from injured corneal epithelial cells could be a mediating factor, either causing keratocyte activation and up-regulation o f KGF and HGF, (Weng J et a l, 1997) and/or by initiating keratocyte apoptosis. (Wilson SE et a l,

1996a) Soluble factors released from regenerating comeal epithelium in conjunction with an intrastromal wound have been shown to induce subepithelial deposits (if the wound is less than 140pm from the surface.) (Baldwin HC et a l, 1999) This supports the hypothesis that epithelial damage results in release o f factors that can stimulate keratocyte activation and scar tissue deposition and that maintaining an intact epithelium during surgery reduces scarring.

MMP-2, mostly in the pro-enzyme form, has been found in the unwounded corneas o f rabbits, rats and humans and may be derived from the aqueous humour. (Fini ME & Girard MT,

1990a) (Ando H et al., 1993) MMP-2 is the main MMP found in remodelling wounds both comeal (Matsubara M et al, 1991a) and cutaneous. (Ashcroft OS et al, 1997) (Neely AN et al, 1999) MMP-1 is necessary for the migration o f kératinocytes on a collagen type I matrix, (Pilcher BK et al., 1997) and is expressed by basal kératinocytes as they migrate across re- epithelialising skin wounds. (Inoue M et al., 1995) Within 24 hours o f corneal wounding by surgical or laser keratectomy (but not epithelial debridement) in rabbits the expression o f MMP-1, -3 and -9 has been found, along with upregulation and activation o f MMP-2.

(Matsubara M et al., 1991a) (Girard MT et al., 1993) (Azar DT et al., 1996) (Azar DT et al.,

1998) (Ye HQ & Azar DT, 1998) MMP-9 was localised to the epithelial basement membrane zone and anterior stroma up to 3 days after wounding, and MMP-2 was predominant in the basal epithelium and superficial stroma at 3 and 7 days after PRK. (Ye HQ & Azar DT, 1998) MMP-7 (Matrilysin) expression was found in migrating basal epithelial cells after PRK in the rat and MMP-3 in deep stromal layers in the first 3 days and in the area o f newly synthesised stromal matrix at 1 week. (Ye HQ & Azar DT, 1998), (Lu PCS et al., 1999) MMP-9 expression coincides with the timing o f new basement membrane synthesis and enzyme loss coincides with the appearance o f new BM components. A role for MMP-9 in re-

epithelialisation and control o f BM resynthesis was suggested rather than long-term stromal re-modelling which is performed by MMP-2. (Matsubara M et al., 1991a)

In this study, corneas were harvested at 4 hours, 18 hours, 24 hours, 3 days, 7 days and 14 days after surgery. Early time points were chosen in order to look at the distribution o f MMPs particularly during the phase o f re-epithelialisation in the SK wounds compared with the non re-epithelialising LK wounds. This study was not designed to look at scar formation and so later time points were not used. MMP-2 is known from previous studies to be involved in stromal remodelling and it may have been interesting to look at the distribution in the 2 wound models at a later time point, for example 3 months. (Matsubara M et al, 1991a) However, animal housing costs and lack o f time precluded a longer time point.

In conclusion, this data shows that MMPs-1 and -9 may be fundamental to the process o f comeal wound healing, particularly epithelial closure and basement membrane formation, confirming previous work in the area. This suggests that it may be possible to use specific MMP inhibitors to modify re-epithelialisation and basement membrane formation after PRK, but that with broad spectmm MMP inhibitors there is a risk o f persistent epithelial defects. Development o f specific inhibitors may provide a promising and directed alternative to ascertain the exact role o f the individual MMPs involved in experimental comeal wound healing as does the development and study o f knockout mice. Work on MMP-9 knockout mice has shown a faster rate o f re-epithelialisation after wounding. (McCabe FJ et a i, 1999) MMP-Is may warrant ftirther investigation as a possible therapy to reduce scarring after comeal surgery.

In document Obtención de aceite de aguacate microencapsulado mediante secado por atomización (página 44-50)

1.3 Microencapsulación de aceites

1.3.4 Secado por aspersión como un proceso para microencapsulación de

1.3.4.5 Selección del material de pared

The distribution o f matrix metalloproteinases 1, 2, 3, and 9

during corneal wound healing

Contents

Summary

Introduction

Methods

Results

Discussion

Summary

Introduction

Methods

Surgical Procedure

Histology and immunohistochemistry

Results

Clinical examination

Histological examination

MMP expression

Superficial keratectomy

Lamellar Keratectomy

%

Superficial

Keratectomy

t f

M M P l

Lamellar

Keratectomy

4 hours

18 hours

24 hours

Superficial

Keratectomy

MMP 1

Lamellar

Keratectomy

3 days

7 days

14 days

x 200

Superficial

Keratectomy

MMP 2

Lamellar

Keratectomy

4 hours

18 hours

24 hours

Superficial

Keratectomy

MMP 2

Lamellar

Keratectomy

3 days

14 days

Superficial

Keratectomy

MMP 3

Lamellar

Keratectomy

4 hours

18 hours

24 hours

Superficial

Keratectomy

MMP 3

Lamellar

Keratectomy

0

3 days

7 days

14 days

Superficial

Keratectomy

MMP 9

Lamellar

Keratectomy

4 hours