Effect of muscarinic agents on sclera fibroblast and their role in myopia

Chick sclera from experimentally myopic and non myopic control eye.. Atropine receiving myopic eye Atropine receiving non myopic control eye Fig3B.. Chick sclera of atropine treated exp

Fig 3

Sclera from myopic eye Sclera from non myopic control eye

Fig3A Chick sclera from experimentally myopic and non myopic control eye

The outer fibrous layer thinned while the inner cartilaginous layer thickened

Atropine receiving myopic eye Atropine receiving non myopic control eye

Fig3B Chick sclera of atropine treated experimentally myopic and control eye

Atropine increased thickness of fibrous layer and decreased cartilaginous layer

The change was towards to a structure of sclera from non myopic eye

Fig 8A Effect of atropine inhibiting SF cell proliferation in culture

Chick SF were incubated with atropine Phase contrast photomicrographs were taken after 24 hrs (magnification x100)

A Control culture without atropine

B Atropine 100 µM caused a significant growth inhibition

The cell morphology appeared to be undisturbed

Fig8B Effect of atropine on DNA integrity (TUNNEL assay)

Chick SF were grown on chamber slides and incubated with atropine (0.1-100 µM) TUNNEL assay was performed after 24 hrs and photomicrographs are taken (magnification x100)

Positive control D Atropine 10 µM

(control culture stimulated to undergo

apotosis produced dark brown stain

in apoptic nuclei)

B Atropine 0.1 µM E Atropine 100 µM

C Atropine 1 µM

Fig10A Immumocytochemistry of mAchR subtypes in human SF in culture Subtype selective antibodies bound to cultured SF demonstrates the presence of m1, m2, m4 receptors (shown in green) When secondary FITC labeled antibody was used without the primary antibody no significant binding was observed (negative control)

m1 m4

m2 negative control

Fig10B Protein corresponding to mAchR subtypes m1,m2,m4, and m5 identified in mouse sclera by immunoblotting Subtype selective antibodies bound to specific protein bands in mouse sclera extract Sepcific bands did not appear in the control experiments, in which the primary antibody is omitted Numbers on left of panels are molecular mass markers (Kd) M1 Negative control

M2 M4

M5

Fig 21A Chick sclera protein extract and SF cell lysates were analysed by gelatin zymography

1 2 3 4

Lane 1 and 2; chick sclera fibroblast cell lysate,

Lane 3 and 4; chick sclera protein extract

Fig19B Westernblot ananlysis of chick sclera protein extract and SF cell lysate using antihuman MMP-2 antibody

1 2 3

Lane 1 ;MMP-2 standard,

Lane 2 ; chick sclera fibroblast cell lysate

Lane 3 ; chick sclera protein extract

Fig19C Western blot analysis of chick sclera protein extract and SF cell lysate using anti human MMP-9 antibody

1 2 3

Lane 1: chick sclera fibroblast cell lysate

Lane 2: chick sclera protein extract

Land 3: MMP-9 standard

Diagram 1 Mechanism of growth factor action and related events

Diagram 2 Interaction between muscarinic and EGF receptors in growth regulation

Diagram 3 Mechanism of G protein coupled m1 receptor