IF recombinant serratiopeptidase derived from Serratia marcescens E-15 (single intrathymic injection, 2.5 mg/kg per lobe, delivered in 5–10 µL sterile vehicle via 33-gauge microsyringe under stereomicroscopic guidance) is administered bilaterally into the thymic parenchyma of 24-month-old male C57BL/6 mice,

THEN immunohistochemical protein density of fibrin, fibronectin, and collagen IV will be reduced by ≥60% relative to aged vehicle controls and restored to within 20% of 3-month-old reference tissue at 7 days post-injection, without exceeding a 15% reduction in total thymic cellularity, AND this ECM clearance will be accompanied by: (a) LC-MS/MS proteomics at 48 hours confirming selective degradation of the target substrates with sparing of laminin and collagen I; (b) reduced TUNEL positivity and increased Ki67 staining in K5+ cortical and K8+ medullary thymic epithelial cell (TEC) populations relative to aged controls; and (c) elevated hydroxyproline content in lavage fluid (not tissue) confirming extracellular collagen IV fragment release rather than structural scaffold destruction,

BECAUSE the following causal chain is supported by the Evidence Set:

1. Serratiopeptidase is a zinc-dependent metalloprotease (~50–60 kDa) with Michaelis-Menten kinetics demonstrating high substrate affinity for fibrin and fibronectin and activity against denatured/pathologically crosslinked collagen IV, while exhibiting substantially reduced activity against structurally intact laminin and collagen I — the proteins that constitute the essential TEC survival scaffold — thereby defining a proteolytic window selective for involuted-thymus-type pathological ECM while sparing normal basement membrane architecture (Tiwari, Asian J Pharm Sci 2017; Biotechnol Appl Biochem 2017, cited in Evidence Set synthesis).

2. The aged C57BL/6 thymic microenvironment (18–24 months) is specifically enriched in exactly these three serratiopeptidase-susceptible proteins — fibrin, fibronectin, and collagen IV — relative to young controls (2–4 months), accumulating preferentially in the perivascular space and interstitium where they displace functional TECs and create a stiff, inhibitory matrix (Clin Exp Immunol 2007; J Immunol 2013, cited in Evidence Set synthesis). This spatial overlap between enzyme-target substrate distribution and pathological ECM accumulation sites makes intrathymic delivery geometrically rational.

3. Direct intrathymic injection of soluble proteins at volumes of 5–20 µL per lobe is technically established in C57BL/6 mice using fine-gauge microsyringes, with diffusion throughout the cortex and medulla confirmed by tracer studies in foundational immunological protocols (Cold Spring Harbor Protocols, doi:10.1101/pdb.prot5366; JoVE protocol, doi:10.3791/2056). Retention and bioavailability of the enzyme within the parenchyma is expected within the relevant time window based on precedents for intrathymic viral protein delivery (doi:10.1371/jou...

SENS category: RepleniSENS