Tracking of In-111-labeled human umbilical tissue-derived cells (hUTC) in a rat model of cerebral ischemia using SPECT imaging

Abstract

Background: In order to increase understanding of how infused cells work, it becomes important to track their initial movement, localization, and engraftment efficiency following transplantation. However, the available in vivo cell tracking techniques are suboptimal. The study objective was to determine the biodistribution of intravenously administered Indium-111 (In-111) oxine labeled human umbilical tissue-derived cells (hUTC) in a rat model of transient middle cerebral occlusion (tMCAo) using single photon emission computed tomography (SPECT).

Methods: Rats received 3 million In-111 labeled hUTC (i.v.) 48 hrs after tMCAo. Following the administration of either hUTC or equivalent dose of In-111-oxine (18.5 MBq), animals underwent SPECT imaging on days 0, 1, and 3. Radioactivity in various organs as well as in the stroke area and contralateral hemisphere was determined, decay corrected and normalized to the total (whole body including head) radioactivity on day 0. Immunohistochemical analysis was also performed to confirm the beneficial effects of hUTC on vascular and synaptic density, and apoptosis.

Results: Most of the radioactivity (43.36±23.07% on day 0) trafficked to the lungs immediately following IV administration of In-111 labeled hUTC (day 0) and decreased drastically to 8.81±7.75 and 4.01±4.52% on days 1 and 3 post-injection, respectively. In contrast, radioactivity measured in the lung of animals that received In-111-oxine alone remained relatively unchanged from day 0 to day 1 (18.38±5.45% at day 0 to 12.59±5.94%) and decreased to 8.34±4.25% on day 3. Significantly higher radioactivity was observed in stroke areas of animals that received In-111 labeled hUTC indicating the presence of cells at the site of injury representing approximately 1% of total administered dose. In addition, there was significant increase in vascular and synaptophysin immunoreactivity in stroke areas of rats that received In-111 labeled hUTC.

Conclusions: The present studies showed the tracking of In-111 labeled hUTC to the sites of stroke in a rat model of tMCAo using SPECT. Animals treated with In-111 labeled hUTC showed histological improvements, with higher vascular and synaptic densities observed in the ischemic boundary zone (IBZ).

Figure 1

Representative images of an animal administered 3 million In-111 labeled hUTC. Most of the cells trafficked to the lungs soon after administration (day 0) and gradually decreased over time with increasing radioactivity observed in the liver. Note the early accumulation of radioactivity in ipsilateral (right side) of the brain (tMCAo side) on day 0. IPL = ipsilateral, CNL = contralateral, R= right side.

Figure 2

Representative images of an animal treated with In-111 oxine alone (vehicle). The radioactivity observed in the lung and liver remained relatively constant over time. Note that there was no preferential increase in the radioactivity in the ipsilateral (right) brain (tMCAo side). IPL = ipsilateral, CNL = contralateral, R = right side.

Figure 3

Biodistribution in the whole body, lungs, liver and kidneys. Relative percent radioactivity in the whole body (A), lungs (B), liver (C) and kidneys (D) of rats that received In-111 oxine vehicle (n=12) or 3 million In-111 labeled hUTC (n=13) 48 hrs after tMCAo. Animals underwent SPECT imaging at day 0, 1 and 3 post-administration and data was normalized to the body radioactivity content on day 0 and corrected for In-111 half-life. WB = whole body, Dc = decay corrected. * = significant differences compared to day 0, # = significant difference between day 1 and 3.

Figure 4

Accumulation of administered In-111 labeled hUTC in the stroke lesion compared to that of In-111 oxine treated animals. Images were from two representative animals in respective groups in In-111 labeled hUTC (top panel) and In-111 oxine vehicle (bottom panel). SPECT images showed higher radioactivity (A) in the lesions (ipsilateral side (dotted circle) and top panel) and the semi-quantitative analysis showed increased in the ipsilateral to contralateral ratio in animals that received In-111 labeled hUTC (n=13) or In-111 oxine vehicle (n=12) (B). * = significant differences compared to corresponding day 0, and # significant differences compared to corresponding In-111-labeled hUTC.

Figure 5

vWF, Synaptophysin, human cell specific staining (anti β2 mitochondrial antibody) and staining for rat macrophages. Upper (first) panel: vWF staining showed significantly higher number of vWF positive cells in animals that received In-111 labeled hUTC. 2nd panel: Similarly synaptophysin staining also showed significantly increased expression in animals that received In-111 hUTC. 3rd panel: A few human specific β-mitochondria positive cells were observed in animals that received In-111 hUTC (black arrows) indicating presence of human cells. Lower (fourth) panel: Sections are from IBZ in animals that received In-111-oxine (left) and In-111-labeled hUTC (right). CD68 positive cells (green color, white arrows) are abundant in IBZ. However, a few human cells (Red color, yellow arrows) are seen in animals that received labeled cells. A few red colored spots are seen in both sections (green arrows), which do not correspond to any nucleus (DAPI positive) are probably artifacts.