Fig. 7: Injection site histopathology.

Following a single IM dose of NM1TFV or NTAF male SD rats were sacrificed on days 7, 28, and 56. a H&E staining of NM1TFV (i) revealed the formulation was readily stained by hematoxylin (ii), but not eosin (iii). For the histological examination of the site of injection, 5 μM sections of paraffin-embedded tissues were stained with hematoxylin and eosin. Images were captured using a Nuance EX multispectral imaging system affixed to a Nikon Eclipse E800 microscope (Nikon Instruments). At day 7, a necrotic focus was observed (i, indicated by *), surrounded by histiocytes, and fibroblasts (ii, arrow). Clusters of foreign material, likely NM1TFV were also observed interspersed in nuclear debris (iii, arrows). By day 28, larger extracellular basophilic staining globules (iii, indicated by *) along with small basophilic intracellular granules present in the histiocytes (iii, indicated by arrow), along with foreign body granulomatous reaction. At 56 days, the foreign material appeared mostly amorphous (i, indicated by *), with a foreign body giant cell granulomatous reaction (ii, iii). b TEM images of the NM1TFV site of injection at day 28 (i & ii), and 56 (iii & iv) confirmed the light microscopic findings. a, b Study was performed once; images are representative of the histological findings from three biological replicates. Magnification of each micrograph is as follows: 1×, 60×, 60× for NM1TFV i, ii, iii, respectively. 4×, 20×, 40× for Day 7 i, ii, iii, respectively. 4×, 10×, 40× for Day 28 i, ii, iii, respectively. 4×, 4×, 40× for Day 56 i, ii, iii, respectively.