Fig 2: STRING protein-protein interaction network of four top up-regulated genes in cluster S4. (A) Protein-protein interaction network connectivity among SPP1, TIMP1, MMP9, and DMP1. In (B, C) protein-protein networks are shown with increasing number of closely associated genes. An edge was drawn with colored lines representing a type of evidence used in predicting the associations. A purple line indicates experimental evidence; a light green line indicates text-mining evidence; a light blue line indicates database evidence; a black line indicates co-expression evidence; and a light violet line indicates protein homology. The score interactions are summarized in Table 1. In (D) UMAP plot with marked cluster S4 enriched in cells expressing Spp1, Timp1, Mmp9, and Dmp1 in response to induction of ERBB2 signaling. In (E) UMAP plots and split violin plots showing up-regulated expression of Spp1, Timp1, Mmp9, and Dmp1 in CA-ERBB2 compared to Control cells in different clusters. (F) RT-qPCR validation of scRNA-seq analysis performed on GFP + FACS-sorted cells from P3 cochleae. The level of gene expression in CA-ERBB2 sample is presented as ??Ct value (± SD) relative to control (CTRL) normalized against the expression of the two reference genes Eef1a1 (E) and Tubb4a (T) (n = 3). Significance (*p < 0.05; **p < 0.01) was determined by unpaired t-test (ONE–TAILED) analysis.

Fig 3: SPP1 and TIMP1 together with CD44 signaling are up-regulated in cochlear cells 2 days after CA-ERBB2 induction in noise exposed young adults. (A) Timeline of noise exposure (NE), GFP activation (TAM), CA-ERBB2 activation (DOX), and hearing tests (HT) in young adult mice. Mice were exposed to 110 dB octave band noise for 2 h, and 3 days later injected with doxycycline (DOX) to induce CA-ERBB2 expression. HT were performed 1 DPN to confirm hearing loss. Immunostaining of cochlea sections revealed detection of SPP1 (B) and TIMP1 (C) together with activated CD44 receptor in CA-ERBB2 animals. Immunodetection for HCs (PVALB, red), and cells competent to express CA-ERBB2 (GFP, green), are shown in panels 1 and 2. Immunodetection of activated CD44 receptor (blue) is shown in panels 1 and 3. Immunodetection of SPP1 and TIMP1 are shown in panel 1, 3, and 4 (gray).

Fig 4: Graphical Summary.Notes: 1. In this study, we formulated TIMP-1 loaded NPs using multiple emulsion solvent evaporation method. The NPs were coated with Ps80 to improve their BBB penetration. 2. We used RBE4/RBCEC as our in vitro BBB model. 3. We compared toxicity of Ps80 coated and non-coated NPs using LY assay, LDH assay, and ZO1 immuno. And we have shown that either of the NPs are nontoxic to endothelial cells. 4. Next, we evaluated the BBB penetration for dye NPs using spectrophotometry and ELISA for TIMP-1 NPs and we have shown that Ps80 coated enhances the in vitro BBB penetration. 5. Finally, we evaluated in vivo BBB penetration by injecting the Ps80 coated TIMP-1 loaded NPs through tail vein injection and it showed that BBB penetration of intravenously injected TIMP-1 NPs + Ps80. 6. To summarize, we have developed TIMP-1 loaded PLGA NPs which can deliver TIMP-1 in a sustained release manner and can cross the BBB. the in vitro and in vivo results have shown that NPs are nontoxic to endothelial cells and they have BBB penetration.Abbreviations: NPs, nanoparticles; TIMP, tissue inhibitor of matrix metalloproteinases; Ps80, polysorbate 80; LDH, lactate dehydrogenase; LY, Lucifer yellow; BBB, blood-brain barrier; ELISA, enzyme-linked immunosorbent assay; immuno, immunocytochemistry; RBE4, rat brain endothelial cell line; RBCEC, rat brain capillary endothelial cells; PLGA, Poly(lactic-co-glycolic acid); ZO1, zona occludens 1.

Fig 5: MMP-9/TIMP-1 relationship in the sciatic nerve. A, Left, Multiple murine MMP-9 species are detected by gelatin zymography. The total extracts (10 µg total protein each) of the naïve (N), distal (D) and proximal (P) segments of the transected nerves were analyzed by gelatin zymography. Right lane, the purified MMP-9 sample isolated from an aliquot (25 µg total protein) of the proximal nerve extract (P) is provided as a reference. Middle, the purified MMP-9 samples isolated from the naïve (N) and crushed (I) nerve (50 µg and 25 µg total protein, respectively) 1, 3, 6 and 24 h post-injury were analyzed by gelatin zymography. Note a 2× protein loading of the naïve samples relative to the injured samples. Right, 2% agarose gel-electrophoresis of the RT-PCR products. The MMP-9 and TIMP-1 genes were amplified using the naive and crushed samples in the RT-PCR reactions. The unique primers were used in a 35-cycle PCR reaction to generate a 153 bp MMP-9 fragment, and 129 bp and 141 bp TIMP-1 fragments. GAPDH was used for sample normalization. Right lane (cDNA), the first strand cDNA control synthesized from the total mouse spleen RNA in the RT-PCR reaction. B, High molecular weight forms of MMP-9 in the injured sciatic nerve. The reduced (+DTT) and unreduced (-DTT) purified MMP-9 samples (15 µg total protein each) isolated from the injured sciatic nerve 0, 1, 3, 6 and 24 h post-injury were analyzed by Western blotting with the MMP-9 antibody. C, Immunofluorescence for MMP-9 and TIMP-1 co-localize in mSCs of crushed nerve 24 h post-injury. Upper panel, MMP-9 (green) and TIMP-1 (red) co-localize in crescent structures of mSCs (arrows). Circular structures are MMP-9 but not TIMP-1 reactive (arrowheads). Lower panel, MMP-9 (red) localizes in the cytoplasm of mSCs, marked with MBP (green). MMP-9 is also detected in axoplasm of mSCs (arrows). Scale bar, 10 µm.