Journal: Investigative Ophthalmology & Visual Science

Article Title: TGFβ Signaling Dysregulation May Contribute to COL4A1-Related Glaucomatous Optic Nerve Damage

doi: 10.1167/iovs.65.5.15

Figure Lengend Snippet: Validation of Cre-mediated recombination of the Tgfbr2 flox allele. ( A ) Genomic structures of Tgfbr2 isoforms with the recombined exon highlighted in the red dashed box . Arrows indicate primer pairs used in B (F1 and R1) or C (F2 and R2). ( B ) Reverse transcription PCR analysis using primers located in common exons (F1 and R1) flanking the floxed exon resulted in amplification of a 553-bp fragment and a 478-bp fragment in the absence of Cre, showing expression of both the long and short isoforms in P7 anterior segments. In the presence of Cre, the floxed exon was excised, resulting in two additional PCR products with smaller sizes (384 bp and 309 bp for the long and short isoforms, respectively); n = 5 Col4a1 +/+ ; Tgfbr2 +/flox samples and n = 7 Col4a1 +/+ ; Tgfbr2 +/ − samples. ( C ) qPCR analysis using a forward primer located in the floxed exon confirmed successful recombination in anterior segments from P7 Col4a1 +/+ and Col4a1 +/G1344D mice. Only wild-type alleles can be amplified using these primers (F2 and R2), and the amount of wild-type mRNA was reduced to ∼50% in mice with Cre expression compared to those without Cre; n = 11 or 12 per genotype. ( D ) Representative images and quantification of Western blots using an antibody recognizing the extracellular domain of TGFBR2 showing reduced TGFBR2 protein levels in the presence of CRE; n = 6 per genotype. ( E ) qPCR analysis of a major TGFβ target gene, Serpine1 , in P7 anterior segments from Col4a1 mice with or without inactivated Tgfbr2 . Serpine1 mRNA levels were increased in Col4a1 +/G1344D ;Tgfbr2 +/flox mice compared to controls ( Col4a1 +/+ ;Tgfbr2 +/flox mice) . The increase was prevented by Tgfbr2 inactivation (comparing Col4a1 +/G1344D ;Tgfbr2 +/flox to Col4a1 +/G1344D ;Tgfbr2 +/ − mice). Two replicates per genotype group are shown in B and D . Data are shown as fold expression relative to Col4a1 +/+ ;Tgfbr2 +/flox and are presented as mean ± SD; n = 10 to 12 per genotype. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001, one-way ANOVA with Sidak's multiple comparison test.

Article Snippet: Membranes were blocked in 10% BSA in Tris-buffered saline with 0.1% Tween 20 (TBST) overnight at 4°C and incubated with rabbit anti-TGFBR2 antibody (AF532-SP, 1:100; R & D Systems, Minneapolis, MN, USA) in 5% BSA in TBST at 4°C for 48 hours.

Techniques: Reverse Transcription, Amplification, Expressing, Western Blot, Comparison

Journal: Investigative Ophthalmology & Visual Science

Article Title: TGFβ Signaling Dysregulation May Contribute to COL4A1-Related Glaucomatous Optic Nerve Damage

doi: 10.1167/iovs.65.5.15

Figure Lengend Snippet: Tgfbr2 heterozygosity reduces ASD in Col4a1 +/G1344D mice. ( A ) Representative slit-lamp images of 1.3- to 1.5-month-old eyes showing examples of mild, moderate, and severe ASD, which typically manifested as dilated and tortuous iris vasculature, open pupil, cataracts, and enlarged anterior chamber. Top panels show frontal views of the eyes; bottom panels show side views of the eyes. White arrows indicate anterior chamber depth. ( B ) Histogram showing the percentage of eyes presenting with mild, moderate, and severe ASD in mice with the indicated genotype; n = 28 to 34 eyes for each genotype. ( C , D ) Representative OCT images of anterior segments ( C ) and quantification of CCT ( D ) showing significantly reduced CCT in 1.6- to 2.0-month-old Col4a1 +/G1344D mice compared to control littermates that was ameliorated by Tgfbr2 heterozygosity (comparing Col4a1 +/G1344D ;Tgfbr2 +/ − to Col4a1 +/G1344D ;Tgfbr2 +/flox mice) . Red bar indicates CCT. C, cornea; I, iris; L, lens. ( E , F ) Representative OCT images of 1.6- to 2.0-month-old eyes ( E ) and quantification of various ocular biometric parameters ( F ) show increased ocular axial length (AL), anterior chamber depth (ACD), lens thickness, and vitreous chamber depth (VCD) in Col4a1 +/G1344D mice compared to controls. Tgfbr2 heterozygosity partially restored AL and VCD and tended to improve ACD and lens diameter in Col4a1 +/G1344D mutant eyes. Blue , yellow , red , and green bars indicate ocular measurements for AL, ACD, lens thickness, and VCD, respectively; n = 26 to 30 eyes for each genotype. Data are presented as mean ± SD. ** P < 0.01; *** P < 0.001; **** P < 0.0001, Fisher's exact test ( B ) and one-way ANOVA with Sidak's multiple comparison test ( D , F ).

Article Snippet: Membranes were blocked in 10% BSA in Tris-buffered saline with 0.1% Tween 20 (TBST) overnight at 4°C and incubated with rabbit anti-TGFBR2 antibody (AF532-SP, 1:100; R & D Systems, Minneapolis, MN, USA) in 5% BSA in TBST at 4°C for 48 hours.

Techniques: Control, Mutagenesis, Comparison

Journal: Investigative Ophthalmology & Visual Science

Article Title: TGFβ Signaling Dysregulation May Contribute to COL4A1-Related Glaucomatous Optic Nerve Damage

doi: 10.1167/iovs.65.5.15

Figure Lengend Snippet: Tgfbr2 heterozygosity ameliorates iridocorneal angle pathology in Col4a1 +/G1344D mice. ( A , B ) Representative H&E-stained ocular sections from 2.0- to 2.5-month-old mice ( A ) and 7- to 9-month-old mice ( B ) showing that, in contrast to the presence of an open iridocorneal angle with identifiable TM ( asterisks ) and Schlemm's canal (SC; bracket ) observed in Col4a1 +/+ ;Tgfbr2 +/flox and Col4a1 +/+ ;Tgfbr2 +/ − eyes, Col4a1 mutant eyes ( Col4a1 +/G1344D ;Tgfbr2 +/ flox and Col4a1 +/G1344D ;Tgfbr2 +/ − ) showed variable degrees of pathology, including partially occluded (compressed TM and SC) or occluded (severe iridocorneal adhesion or ICA; black arrows ). In addition, the ciliary body was often small and unfoliated ( open arrowheads ). Scale bar : 100 µm. ( C ) Frequency of open, partially occluded, or occluded angles with indicated genotype; n = 16 Col4a1 +/+ ;Tgfbr2 +/flox , n = 15 Col4a1 +/+ ;Tgfbr2 +/ − , n = 33 Col4a1 +/G1344D ;Tgfbr2 +/flox , and n = 19 Col4a1 +/G1344D ;Tgfbr2 +/ − angles at 2.0 to 2.5 months; n = 20 Col4a1 +/+ ;Tgfbr2 +/flox , n = 24 Col4a1 +/+ ;Tgfbr2 +/ − , n = 24 Col4a1 +/G1344D ;Tgfbr2 +/flox , and n = 18 Col4a1 +/G1344D ;Tgfbr2 +/ − angles at 7 to 9 months. ( D ) Quantification of ICA length showing that ICA worsens with ages in Col4a1 +/G1344D eyes and can be partially prevented by Tgfbr2 heterozygosity; n = 25 Col4a1 +/G1344D ;Tgfbr2 +/flox and n = 12 Col4a1 +/G1344D ;Tgfbr2 +/ − angles at 2.0 to 2.5 months; n = 19 Col4a1 +/G1344D ;Tgfbr2 +/flox and n = 12 Col4a1 +/G1344D ;Tgfbr2 +/ − angles at 7 to 9 months. Data are presented as mean ± SD. * P < 0.05; *** P < 0.001, Fisher's exact test ( C ) and Student's t -test with Welch's correction ( D ).

Article Snippet: Membranes were blocked in 10% BSA in Tris-buffered saline with 0.1% Tween 20 (TBST) overnight at 4°C and incubated with rabbit anti-TGFBR2 antibody (AF532-SP, 1:100; R & D Systems, Minneapolis, MN, USA) in 5% BSA in TBST at 4°C for 48 hours.

Techniques: Staining, Mutagenesis

Journal: Investigative Ophthalmology & Visual Science

Article Title: TGFβ Signaling Dysregulation May Contribute to COL4A1-Related Glaucomatous Optic Nerve Damage

doi: 10.1167/iovs.65.5.15

Figure Lengend Snippet: Tgfbr2 heterozygosity affects IOP in Col4a1 +/G1344D mice. ( A , B ) IOP measurements in mice at 2.0 to 2.5 months ( A ) and at 7 to 9 months ( B ). Multiple Col4a1 +/G1344D eyes had high IOP (>21 mmHg) at both ages, and at 7 to 9 months the Col4a1 +/G1344D eyes showed higher average IOPs than Col4a1 +/+ eyes. Tgfbr2 heterozygosity at 7 to 9 months did not affect the mean of IOPs in Col4a1 +/G1344D eyes, although the variation was smaller and the incidence of eyes with high IOP was reduced; n = 14 and 22 Col4a1 +/+ ;Tgfbr2 +/flox eyes, n = 23 and 20 Col4a1 +/+ ;Tgfbr2 +/ − eyes, n = 21 and 9 Col4a1 +/G1344D ;Tgfbr2 +/flox eyes, and n = 21 and 10 Col4a1 +/G1344D ;Tgfbr2 +/ − eyes at 2.0 to 2.5 months and at 7 to 9 months of age, respectively. Dashed line indicates IOP = 21 mmHg. Data are presented as mean ± SD. * P < 0.05, one-way ANOVA with Sidak's multiple comparison test.

Article Snippet: Membranes were blocked in 10% BSA in Tris-buffered saline with 0.1% Tween 20 (TBST) overnight at 4°C and incubated with rabbit anti-TGFBR2 antibody (AF532-SP, 1:100; R & D Systems, Minneapolis, MN, USA) in 5% BSA in TBST at 4°C for 48 hours.

Techniques: Comparison

Journal: Investigative Ophthalmology & Visual Science

Article Title: TGFβ Signaling Dysregulation May Contribute to COL4A1-Related Glaucomatous Optic Nerve Damage

doi: 10.1167/iovs.65.5.15

Figure Lengend Snippet: Tgfbr2 heterozygosity reduced the frequency of optic nerve head excavation and improved retinal thickness in Col4a1 +/G1344D mice. ( A ) Representative OCT images showing the central retina and optic nerve head in mice at 1.6 to 1.8 months and at 7 to 9 months. Although the optic nerve head appeared to be flat in wild-type eyes, Col4a1 +/G1344D eyes showed optic nerve head excavation ( asterisks ) with variable severity. Blue , red , and yellow bars indicate ocular measurements for total retinal, GCC, and ONL thickness, respectively. A magnified image is also shown in . ( B ) Frequency of normal, mildly, or deeply excavated optic nerve heads in wild-type or Col4a1 +/G1344D mice with or without Tgfbr2 heterozygosity at 1.6 to 1.8 months or 7 to 9 months of age. The incidence of severe cupping increased with age in Col4a1 +/G1344D eyes, and it was reduced with Tgfbr2 heterozygosity. ONH, optic nerve head. ( C , D ) Quantification of thickness of different retinal layers by OCT biometry. In both age groups, Col4a1 +/G1344D eyes had thinner GCC layers, reduced total retinal thickness, and thinner outer nuclear layer (ONL). When Tgfbr2 was inactivated, the thickness of those layers was partially restored (except the ONL in the young age group); n = 17 and 21 Col4a1 +/+ ;Tgfbr2 +/flox eyes, n = 14 and 18 Col4a1 +/+ ;Tgfbr2 +/ − eyes, n = 24 and 12 Col4a1 +/G1344D ;Tgfbr2 +/flox eyes, and n = 14 and 11 Col4a1 +/G1344D ;Tgfbr2 +/ − eyes at 1.6 to 1.8 months and 7 to 9 months of age, respectively. Data are presented as mean ± SD. * P < 0.05; ** P < 0.1; *** P < 0.001; **** P < 0.0001, Fisher's exact test ( B ) and one-way ANOVA with Sidak's multiple comparison test ( C , D ).

Article Snippet: Membranes were blocked in 10% BSA in Tris-buffered saline with 0.1% Tween 20 (TBST) overnight at 4°C and incubated with rabbit anti-TGFBR2 antibody (AF532-SP, 1:100; R & D Systems, Minneapolis, MN, USA) in 5% BSA in TBST at 4°C for 48 hours.

Techniques: Comparison

Journal: Investigative Ophthalmology & Visual Science

Article Title: TGFβ Signaling Dysregulation May Contribute to COL4A1-Related Glaucomatous Optic Nerve Damage

doi: 10.1167/iovs.65.5.15

Figure Lengend Snippet: Histological analyses of the retina revealed a protective role of Tgfbr2 heterozygosity in Col4a1 +/G1344D mice. ( A , B ) Representative histological images of the central retina in Col4a1 +/+ or Col4a1 +/G1344D mice with or without Tgfbr2 heterozygosity at 2.0 to 2.5 months of age ( A ) or 7 to 9 months of age ( B ). Black arrowheads indicate the NFLs, and open arrowheads indicate cell bodies in the RGC layer. Although some Col4a1 +/G1344D eyes appeared to be normal with a robust NFL and a continuous layer of cells, others showed thin NFLs accompanied with cell loss. Scale bar : 100 µm. ( C ) Frequency of eyes with thin NFLs and cell loss in mice with indicated genotype and age. Although all control retinas were healthy, approximately 50% of the Col4a1 +/G1344D ;Tgfbr2 +/flox eyes had thin NFLs and discontinued retinal ganglion cells. Tgfbr2 heterozygosity appeared to reduce the frequency of eyes with thin NFLs and cell loss in both age groups. ( D ) Quantification of the NFL thickness. We observed a trend toward protection in mice with Tgfbr2 heterozygosity at 7 to 9 months of age; n = 6 and 10 Col4a1 +/+ ;Tgfbr2 +/flox eyes, n = 6 and 11 Col4a1 +/+ ;Tgfbr2 +/ − eyes, n = 18 and 10 Col4a1 +/G1344D ;Tgfbr2 +/flox eyes, and n = 10 and 8 Col4a1 +/G1344D ;Tgfbr2 +/ − eyes at 2.0 to 2.5 months and 7 to 9 months of age, respectively. *** P < 0.001, Fisher's exact test ( C ) and Kruskal–Wallis test with Dunn's multiple comparison test ( D ).

Article Snippet: Membranes were blocked in 10% BSA in Tris-buffered saline with 0.1% Tween 20 (TBST) overnight at 4°C and incubated with rabbit anti-TGFBR2 antibody (AF532-SP, 1:100; R & D Systems, Minneapolis, MN, USA) in 5% BSA in TBST at 4°C for 48 hours.

Techniques: Control, Comparison

Journal: Investigative Ophthalmology & Visual Science

Article Title: TGFβ Signaling Dysregulation May Contribute to COL4A1-Related Glaucomatous Optic Nerve Damage

doi: 10.1167/iovs.65.5.15

Figure Lengend Snippet: Tgfbr2 heterozygosity prevents optic nerve head damage in Col4a1 +/G1344D mice. ( A , B ) Representative images of H&E-stained ocular sections from mice at 2.0 to 2.5 months ( A ) and at 7 to 9 months ( B ). Although some Col4a1 +/G1344D eyes had a robust NFL and normal optic nerve head morphology, the others showed a thin NFL and tissue loss in the optic nerve head. Black arrowheads indicate the NFL, and the asterisk indicates optic nerve head excavation. Scale bar : 100 µm. ( C ) Frequency of normal, mildly, or deeply excavated optic nerve heads in mice with indicated genotype and age. Tgfbr2 heterozygosity appeared to reduce the frequency of eyes with optic nerve head excavation at both ages examined, and a trend toward significance was observed at 7 to 9 months of age. ONH, optic nerve head; n = 6 and 10 Col4a1 +/+ ;Tgfbr2 +/flox eyes, n = 6 and 11 Col4a1 +/+ ;Tgfbr2 +/ − eyes, n = 18 and 10 Col4a1 +/G1344D ;Tgfbr2 +/flox eyes, and n = 10 and 8 Col4a1 +/G1344D ;Tgfbr2 +/ − eyes at 2.0 to 2.5 months and 7 to 9 months of age, respectively. Fisher's exact test for C .

Article Snippet: Membranes were blocked in 10% BSA in Tris-buffered saline with 0.1% Tween 20 (TBST) overnight at 4°C and incubated with rabbit anti-TGFBR2 antibody (AF532-SP, 1:100; R & D Systems, Minneapolis, MN, USA) in 5% BSA in TBST at 4°C for 48 hours.

Techniques: Staining

Journal: Investigative Ophthalmology & Visual Science

Article Title: TGFβ Signaling Dysregulation May Contribute to COL4A1-Related Glaucomatous Optic Nerve Damage

doi: 10.1167/iovs.65.5.15

Figure Lengend Snippet: Tgfbr2 heterozygosity partially prevents axonal degeneration in optic nerves from Col4a1 +/G1344D mice. ( A , B ) Representative cross-sections of PPD-stained optic nerves from mice at 2.0 to 2.5 months ( A ) or 7 to 9 months ( B ). ( C , D ) Quantification of cross-sectional area ( C ) and total axon number ( D ) showing that, in contrast to the healthy myelinated axons observed in wild-type optic nerves, Col4a1 +/G1344D nerves contained numerous degenerated axons, as indicated by darkly stained axoplasm, and that Tgfbr2 heterozygosity partially prevented axonal loss in Col4a1 +/G1344D optic nerves; n = 8 and 8 Col4a1 +/+ ;Tgfbr2 +/flox eyes, n = 8 and 8 Col4a1 +/+ ;Tgfbr2 +/ − eyes, n = 11 and 11 Col4a1 +/G1344D ;Tgfbr2 +/flox eyes, and n = 11 and 8 Col4a1 +/G1344D ;Tgfbr2 +/ − eyes at 2.0 to 2.5 months and at 7 to 9 months of age, respectively. Scale bar : 20 µm. Data are presented as mean ± SD. * P < 0.05; ** P < 0.1; *** P < 0.001; **** P < 0.0001, one-way ANOVA with Sidak's multiple comparison test.

Article Snippet: Membranes were blocked in 10% BSA in Tris-buffered saline with 0.1% Tween 20 (TBST) overnight at 4°C and incubated with rabbit anti-TGFBR2 antibody (AF532-SP, 1:100; R & D Systems, Minneapolis, MN, USA) in 5% BSA in TBST at 4°C for 48 hours.

Techniques: Staining, Comparison

Journal: The Journal of Physiology

Article Title: Comparison of inhibitory neuromuscular transmission in the Cynomolgus monkey IAS and rectum: special emphasis on differences in purinergic transmission

doi: 10.1113/JP275437

Figure Lengend Snippet: Antibodies

Article Snippet: A list of the primary antibodies is given in Table . table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Primary antibody Source Host Working dilution Anti‐human PDGFRα R&D Systems, Minneapolis, MN, USA Goat 1:100 Anti‐human SCFR (Kit) R&D Systems Goat 1:100 Anti‐nNOS (used with anti‐Kit and anti‐PDGFRα) Santa Cruz Biotechnology Inc., Dallas, TX, USA Rabbit 1:500 Anti‐nNOS (used with anti‐VIP) Gift from Dr Piers Emson, Molecular Science Group, Cambridge, UK Sheep 1:500 Anti‐SK3 Alomone Labs, Jerusalem, Israel Rabbit 1:500 Anti‐VIP Immunostar Antibodies, Hudson, WI, USA Rabbit 1:500 Open in a separate window Antibodies Imaging Cryosections were examined using a Zeiss LSM 510 confocal microscope (Carl Zeiss, Thornwood, NY, USA).

Techniques: