pgp9 5  (Bio-Rad)

Journal: bioRxiv

Article Title: 3D Light Sheet Microscopy Reveals Aging Osteoarthritis-associated Joint-innervated Nerve Remodeling in Mouse Knee Joints

doi: 10.64898/2026.01.09.698640

Figure Lengend Snippet: Male mice experience more severe age-associated OA relative to female mice. Age associated changes in osteoarthritis severity, pain sensitivity and expression of CGRP and PIEZO2 in the DRGs. (A) Schematic of experimental design. (B) Representative sagittal sections stained with Safranin-O/Fast-Green. Blue arrows point degenerated articular cartilage. Scale bars, 200 µm. (C) OARSI scores of the articular cartilage (n = 5-7 mice/group). (D) Quantitative analysis of knee hyperalgesia using the Pressure application measurement (PAM) (n = 5-7 mice/group). (E) Representative immunofluorescent images of PGP9.5, CGRP and PIEZO2 staining in L3-L5 DRGs. Scale bar, 100 μm. (F) CGRP + neuron counts (n = 4-5 mice/group) and (G) anti-Piezo2 relative staining intensity (n = 4-5 mice/group) in DRG sections. Mean ± 95% CI. OARSI and PAM data were analyzed using a non-parametric Kruskal–Wallis test followed by Dunn’s multiple-comparison post hoc test. CGRP and Piezo2 data were analyzed using two-way ANOVA with sex and age as factors followed by uncorrected Fisher’s LSD post hoc tests.

Article Snippet: Given that CGRP is a secreted neuropeptide and is also present in vascular smooth muscle surrounding blood vessels, the complete nerve network was first reconstructed in 3D using PGP9.5+ filaments in Imaris (v10.1.1).

Techniques: Expressing, Staining, Comparison

Journal: bioRxiv

Article Title: 3D Light Sheet Microscopy Reveals Aging Osteoarthritis-associated Joint-innervated Nerve Remodeling in Mouse Knee Joints

doi: 10.64898/2026.01.09.698640

Figure Lengend Snippet: Age-associated increase in total knee joint innervation in male mice. (A) Representative three-dimensional (3D) images of knee joints labeled with anti-PGP9.5 (green) and the reconstructed µCT scans. Quantified regions are shown in yellow (x-y axis) and orange (y-z axis) boxes. (B) Quantification of the imaging coordinate length of the knee joint in young and aged male and female mice respectively in the x-axis, y-axis and z-depth. (C) Representative 3D anti-PGP9.5 (green) micrographs of the knee joint of My (young male), Ma (aged male), Fy (young female) and Fa (aged female); white line traces PGP9.5+ nerve filaments using Imaris v10.1.1 software. (D) Representative MATLAB-generated images showing the boundary volume (gray) encapsulating the PGP9.5+ segment coordinates (red) in My, Ma, Fy and Fa (E) Quantification of the total boundary volume, (F) PGP9.5+ filament length, (G) PGP9.5+ filament length/total boundary volume, (H) PGP9.5+ branch points, and (I) PGP9.5+ branch points/total filament length in young and aged male and female mice, respectively. N = 5 mice/group. Data were analyzed using two-way ANOVA to assess the effects of age and sex, followed by uncorrected Fisher’s LSD post hoc tests. Data are presented as mean ± 95%CI.

Article Snippet: Given that CGRP is a secreted neuropeptide and is also present in vascular smooth muscle surrounding blood vessels, the complete nerve network was first reconstructed in 3D using PGP9.5+ filaments in Imaris (v10.1.1).

Techniques: Labeling, Imaging, Software, Generated

Journal: bioRxiv

Article Title: 3D Light Sheet Microscopy Reveals Aging Osteoarthritis-associated Joint-innervated Nerve Remodeling in Mouse Knee Joints

doi: 10.64898/2026.01.09.698640

Figure Lengend Snippet: Change in CGRP⁺/PGP9.5⁺ nociceptive nerve innervation in the aging knee joints. (A) (top) Representative 3D Light-Sheet Micrographs of knee-innervating nerve fibers stained with anti-PGP9.5 (green) and anti-CGRP (red) in M y , M a , F y and F a ; CGRP + nerve fibers are traced using IMARIS. CGRP⁺ nerve innervation was quantified by first segmenting total nerve fibers using PGP9.5 immunolabeling. A single, fixed intensity threshold was then applied uniformly across all samples to identify CGRP⁺ signal within the PGP9.5⁺ nerve segments to quantify CGRP + PGP9.5 + nerve filament. (B) Total length of CGRP + PGP9.5 + nerve filament. (C) Total CGRP + PGP9.5 + nerve filament length/boundary volume. (D) Total branch points in CGRP + PGP9.5 + nerve filaments. (E) CGRP + PGP9.5 + branch points/total CGRP + PGP9.5 + filament length. N = 3 mice/group.

Article Snippet: Given that CGRP is a secreted neuropeptide and is also present in vascular smooth muscle surrounding blood vessels, the complete nerve network was first reconstructed in 3D using PGP9.5+ filaments in Imaris (v10.1.1).

Techniques: Staining, Immunolabeling

Journal: bioRxiv

Article Title: 3D Light Sheet Microscopy Reveals Aging Osteoarthritis-associated Joint-innervated Nerve Remodeling in Mouse Knee Joints

doi: 10.64898/2026.01.09.698640

Figure Lengend Snippet: Orthogonal planes of view (coronal, sagittal and transverse) from PGP9.5/CGRP immunolabeled knee joints from (A) young male and (B) aged male mice, and from PGP9.5/TH immunolabeled knee joints from (C) young male and (D) aged male mice, depicting the depth of nerve infiltration into the various tissues of the knee joint. Insets capture the femur (inset B), infrapatellar fat pad (inset C), and joint capsule/synovium (inset D). CGRP+ sensory nerves (panels A,B) and TH+ sympathetic nerves (panels C,D) appear to innervate the extrasynovial fat and joint capsule (insets C,D), whereas minimal staining is seen in the femoral bone (inset B), which likely reflects insufficient antibody penetration into bone. White insets are 300 μm x 300 μm.

Article Snippet: Given that CGRP is a secreted neuropeptide and is also present in vascular smooth muscle surrounding blood vessels, the complete nerve network was first reconstructed in 3D using PGP9.5+ filaments in Imaris (v10.1.1).

Techniques: Immunolabeling, Staining

Journal: bioRxiv

Article Title: 3D Light Sheet Microscopy Reveals Aging Osteoarthritis-associated Joint-innervated Nerve Remodeling in Mouse Knee Joints

doi: 10.64898/2026.01.09.698640

Figure Lengend Snippet: Spatial mapping of segment positions in the medial and lateral compartments of the knee joint in young and aged male mice (M y , M a ). (A) k-means clustering was performed in Matlab to partition the segment position data into medial and lateral clusters for PGP9.5, CGRP and TH innervation. The percentage of segments in medial and lateral clusters was quantified for PGP9.5 (n = 5 mice/group) (B) CGRP (n = 3 mice/group) (C), TH (n = 4 mice/group) (D). Medial-lateral proportions for individual samples are connected by lines, with bars representing the mean of each compartment. Repeated measures two-way ANOVA with post-hoc Fisher’s Least Significant Difference (LSD) test was performed to statistically compare differences in medial-lateral nerve proportions across young and aged male mice, matching compartments from individual mice.

Article Snippet: Given that CGRP is a secreted neuropeptide and is also present in vascular smooth muscle surrounding blood vessels, the complete nerve network was first reconstructed in 3D using PGP9.5+ filaments in Imaris (v10.1.1).

Techniques:

Journal: bioRxiv

Article Title: 3D Light Sheet Microscopy Reveals Aging Osteoarthritis-associated Joint-innervated Nerve Remodeling in Mouse Knee Joints

doi: 10.64898/2026.01.09.698640

Figure Lengend Snippet: Spatial mapping of segment positions in the medial and lateral compartments of the knee joint in aged female mice with osteoarthritis compared to young female mice. (A) k-means clustering was performed in Matlab to partition the segment position data into medial and lateral clusters for PGP9.5, CGRP and TH innervation. The percentage of segments in medial and lateral clusters was quantified for PGP9.5 (n = 5 mice/group) (B) CGRP (n = 3 mice/group) (C) TH (n = 4 mice/group) (D) Medial-lateral proportions for individual samples are connected by lines, with bars representing the mean of each compartment. Repeated measures two-way ANOVA with post-hoc Fisher’s Least Significant Difference (LSD) test was performed to statistically compare differences in medial-lateral nerve proportions across young and aged female mice, matching compartments from individual mice.

Article Snippet: Given that CGRP is a secreted neuropeptide and is also present in vascular smooth muscle surrounding blood vessels, the complete nerve network was first reconstructed in 3D using PGP9.5+ filaments in Imaris (v10.1.1).

Techniques:

Journal: bioRxiv

Article Title: 3D Light Sheet Microscopy Reveals Aging Osteoarthritis-associated Joint-innervated Nerve Remodeling in Mouse Knee Joints

doi: 10.64898/2026.01.09.698640

Figure Lengend Snippet: Male mice experience more severe age-associated OA relative to female mice. Age associated changes in osteoarthritis severity, pain sensitivity and expression of CGRP and PIEZO2 in the DRGs. (A) Schematic of experimental design. (B) Representative sagittal sections stained with Safranin-O/Fast-Green. Blue arrows point degenerated articular cartilage. Scale bars, 200 µm. (C) OARSI scores of the articular cartilage (n = 5-7 mice/group). (D) Quantitative analysis of knee hyperalgesia using the Pressure application measurement (PAM) (n = 5-7 mice/group). (E) Representative immunofluorescent images of PGP9.5, CGRP and PIEZO2 staining in L3-L5 DRGs. Scale bar, 100 μm. (F) CGRP + neuron counts (n = 4-5 mice/group) and (G) anti-Piezo2 relative staining intensity (n = 4-5 mice/group) in DRG sections. Mean ± 95% CI. OARSI and PAM data were analyzed using a non-parametric Kruskal–Wallis test followed by Dunn’s multiple-comparison post hoc test. CGRP and Piezo2 data were analyzed using two-way ANOVA with sex and age as factors followed by uncorrected Fisher’s LSD post hoc tests.

Article Snippet: For the filament normalization, the 3D rendered PGP9.5+ nerve network defined in Imaris was imported to MATLAB, where computational geometry was used to calculate the volume of 3D boundary regions enveloping the PGP9.5+ nerve segments ( ).

Techniques: Expressing, Staining, Comparison

Journal: bioRxiv

Article Title: 3D Light Sheet Microscopy Reveals Aging Osteoarthritis-associated Joint-innervated Nerve Remodeling in Mouse Knee Joints

doi: 10.64898/2026.01.09.698640

Figure Lengend Snippet: Age-associated increase in total knee joint innervation in male mice. (A) Representative three-dimensional (3D) images of knee joints labeled with anti-PGP9.5 (green) and the reconstructed µCT scans. Quantified regions are shown in yellow (x-y axis) and orange (y-z axis) boxes. (B) Quantification of the imaging coordinate length of the knee joint in young and aged male and female mice respectively in the x-axis, y-axis and z-depth. (C) Representative 3D anti-PGP9.5 (green) micrographs of the knee joint of My (young male), Ma (aged male), Fy (young female) and Fa (aged female); white line traces PGP9.5+ nerve filaments using Imaris v10.1.1 software. (D) Representative MATLAB-generated images showing the boundary volume (gray) encapsulating the PGP9.5+ segment coordinates (red) in My, Ma, Fy and Fa (E) Quantification of the total boundary volume, (F) PGP9.5+ filament length, (G) PGP9.5+ filament length/total boundary volume, (H) PGP9.5+ branch points, and (I) PGP9.5+ branch points/total filament length in young and aged male and female mice, respectively. N = 5 mice/group. Data were analyzed using two-way ANOVA to assess the effects of age and sex, followed by uncorrected Fisher’s LSD post hoc tests. Data are presented as mean ± 95%CI.

Article Snippet: For the filament normalization, the 3D rendered PGP9.5+ nerve network defined in Imaris was imported to MATLAB, where computational geometry was used to calculate the volume of 3D boundary regions enveloping the PGP9.5+ nerve segments ( ).

Techniques: Labeling, Imaging, Software, Generated

Journal: bioRxiv

Article Title: 3D Light Sheet Microscopy Reveals Aging Osteoarthritis-associated Joint-innervated Nerve Remodeling in Mouse Knee Joints

doi: 10.64898/2026.01.09.698640

Figure Lengend Snippet: Change in CGRP⁺/PGP9.5⁺ nociceptive nerve innervation in the aging knee joints. (A) (top) Representative 3D Light-Sheet Micrographs of knee-innervating nerve fibers stained with anti-PGP9.5 (green) and anti-CGRP (red) in M y , M a , F y and F a ; CGRP + nerve fibers are traced using IMARIS. CGRP⁺ nerve innervation was quantified by first segmenting total nerve fibers using PGP9.5 immunolabeling. A single, fixed intensity threshold was then applied uniformly across all samples to identify CGRP⁺ signal within the PGP9.5⁺ nerve segments to quantify CGRP + PGP9.5 + nerve filament. (B) Total length of CGRP + PGP9.5 + nerve filament. (C) Total CGRP + PGP9.5 + nerve filament length/boundary volume. (D) Total branch points in CGRP + PGP9.5 + nerve filaments. (E) CGRP + PGP9.5 + branch points/total CGRP + PGP9.5 + filament length. N = 3 mice/group.

Article Snippet: For the filament normalization, the 3D rendered PGP9.5+ nerve network defined in Imaris was imported to MATLAB, where computational geometry was used to calculate the volume of 3D boundary regions enveloping the PGP9.5+ nerve segments ( ).

Techniques: Staining, Immunolabeling

Journal: bioRxiv

Article Title: 3D Light Sheet Microscopy Reveals Aging Osteoarthritis-associated Joint-innervated Nerve Remodeling in Mouse Knee Joints

doi: 10.64898/2026.01.09.698640

Figure Lengend Snippet: Orthogonal planes of view (coronal, sagittal and transverse) from PGP9.5/CGRP immunolabeled knee joints from (A) young male and (B) aged male mice, and from PGP9.5/TH immunolabeled knee joints from (C) young male and (D) aged male mice, depicting the depth of nerve infiltration into the various tissues of the knee joint. Insets capture the femur (inset B), infrapatellar fat pad (inset C), and joint capsule/synovium (inset D). CGRP+ sensory nerves (panels A,B) and TH+ sympathetic nerves (panels C,D) appear to innervate the extrasynovial fat and joint capsule (insets C,D), whereas minimal staining is seen in the femoral bone (inset B), which likely reflects insufficient antibody penetration into bone. White insets are 300 μm x 300 μm.

Article Snippet: For the filament normalization, the 3D rendered PGP9.5+ nerve network defined in Imaris was imported to MATLAB, where computational geometry was used to calculate the volume of 3D boundary regions enveloping the PGP9.5+ nerve segments ( ).

Techniques: Immunolabeling, Staining

Journal: bioRxiv

Article Title: 3D Light Sheet Microscopy Reveals Aging Osteoarthritis-associated Joint-innervated Nerve Remodeling in Mouse Knee Joints

doi: 10.64898/2026.01.09.698640

Figure Lengend Snippet: Spatial mapping of segment positions in the medial and lateral compartments of the knee joint in young and aged male mice (M y , M a ). (A) k-means clustering was performed in Matlab to partition the segment position data into medial and lateral clusters for PGP9.5, CGRP and TH innervation. The percentage of segments in medial and lateral clusters was quantified for PGP9.5 (n = 5 mice/group) (B) CGRP (n = 3 mice/group) (C), TH (n = 4 mice/group) (D). Medial-lateral proportions for individual samples are connected by lines, with bars representing the mean of each compartment. Repeated measures two-way ANOVA with post-hoc Fisher’s Least Significant Difference (LSD) test was performed to statistically compare differences in medial-lateral nerve proportions across young and aged male mice, matching compartments from individual mice.

Article Snippet: For the filament normalization, the 3D rendered PGP9.5+ nerve network defined in Imaris was imported to MATLAB, where computational geometry was used to calculate the volume of 3D boundary regions enveloping the PGP9.5+ nerve segments ( ).

Techniques:

Journal: bioRxiv

Article Title: 3D Light Sheet Microscopy Reveals Aging Osteoarthritis-associated Joint-innervated Nerve Remodeling in Mouse Knee Joints

doi: 10.64898/2026.01.09.698640

Figure Lengend Snippet: Spatial mapping of segment positions in the medial and lateral compartments of the knee joint in aged female mice with osteoarthritis compared to young female mice. (A) k-means clustering was performed in Matlab to partition the segment position data into medial and lateral clusters for PGP9.5, CGRP and TH innervation. The percentage of segments in medial and lateral clusters was quantified for PGP9.5 (n = 5 mice/group) (B) CGRP (n = 3 mice/group) (C) TH (n = 4 mice/group) (D) Medial-lateral proportions for individual samples are connected by lines, with bars representing the mean of each compartment. Repeated measures two-way ANOVA with post-hoc Fisher’s Least Significant Difference (LSD) test was performed to statistically compare differences in medial-lateral nerve proportions across young and aged female mice, matching compartments from individual mice.

Article Snippet: For the filament normalization, the 3D rendered PGP9.5+ nerve network defined in Imaris was imported to MATLAB, where computational geometry was used to calculate the volume of 3D boundary regions enveloping the PGP9.5+ nerve segments ( ).

Techniques: