Trauma-hemorrhagic shock (HS) often leads to remote organ injury, particularly the gut and kidney. While fluid resuscitation is essential, optimal strategies to mitigate organ damage remain lacking. This study investigated the combined effects of hypertonic saline (HTS) and ulinastatin (UTI) on HS-induced intestinal and renal injuries. Male Sprague-Dawley rats were randomized into five groups: Sham, HS, HS+HTS, HS+UTI, and HS+HTS-UTI. A standardized HS model was used. Interventions were administered during resuscitation. Hemodynamics were monitored. At 24 h post-resuscitation, serum and tissues (ileum, kidney) were collected. Intestinal and renal injuries were assessed via histopathology, apoptosis (TUNEL), inflammation (ELISA for TNF-α, IL-6), oxidative stress (MDA, SOD), barrier function (FITC-dextran, tight junction proteins ZO-1/Occludin), and renal function/injury markers (creatinine, BUN, KIM-1, NGAL). HS induced severe hypotension, electrolyte disturbance, systemic inflammation, oxidative stress, and histopathological damage in the intestine and kidney, along with increased intestinal permeability, apoptosis, and renal dysfunction. Monotherapy with either HTS or UTI provided partial protection. However, the combination therapy demonstrated superior protective effects, which were significantly greater than either monotherapy alone. The HTS and UTI combination most effectively restored mean arterial pressure, suppressed systemic and tissue inflammation/oxidative stress, preserved intestinal morphology and barrier integrity, reduced intestinal and renal apoptosis, and ameliorated renal histology and function while downregulating KIM-1 and NGAL expression. The mechanism involves simultaneous mitigation of inflammation, oxidative stress, and apoptosis, along with preservation of intestinal barrier and renal tubular integrity.
Quantitative and semiquantitative assessment of biomarker expression using immunohistochemistry (IHC) can be performed using several analytical approaches. Manual counting remains widely used but is inherently subjective and susceptible to observer variability. Rule-based image-analysis platforms may provide accessible alternatives, highlighting the need for direct method-comparison and agreement studies. c-Fos is a well-established marker of neuronal activation commonly quantified by counting IHC-positive cells. This study compared manual counting, ImageJ, and CellProfiler for quantifying c-Fos-positive cells in rat brainstem sections. Of 125 images initially evaluated, one was excluded as an extreme outlier, leaving 124 images for final analysis. Each image was independently analyzed by two trained observers using all three methods. ImageJ and CellProfiler were applied through their standard graphical interfaces and built-in functions using predefined user-guided threshold ranges, without additional plugins, scripts, machine-learning models, or custom code. Agreement was assessed using intraclass correlation coefficients (ICCs) and Bland-Altman analysis. No statistically significant difference in c-Fos-positive cell counts was observed among the three methods (Friedman test, P = 0.07). Median (IQR) counts were 6.75 (0.50-13.50) for manual counting, 6.25 (1.00-14.38) for ImageJ, and 6.00 (0.63-14.50) for CellProfiler. Inter-observer agreement was excellent for manual counting (ICC = 0.983), ImageJ (ICC = 0.964), and CellProfiler (ICC = 0.962). Inter-method agreement was also excellent (single-measures ICC = 0.971; 95% CI = 0.961-0.979). Bland-Altman analysis showed small mean biases of 0.33, 0.27, and -0.07 cells, with no significant proportional bias. Manual counting, ImageJ, and CellProfiler produced statistically comparable c-Fos-positive cell counts with excellent agreement. Under the predefined user-guided threshold ranges used, both software platforms closely matched manual counting; however, image-dependent threshold adjustment remains a potential source of observer variability.
