Laser system refinements to reduce variability in infarct size in the rat photothrombotic stroke model

Abstract

Background: The rat photothrombotic stroke model can induce brain infarcts with reasonable biological variability. Nevertheless, we observed unexplained high inter-individual variability despite using a rigorous protocol. Of the three major determinants of infarct volume, photosensitive dye concentration and illumination period were strictly controlled, whereas undetected fluctuation in laser power output was suspected to account for the variability.

New method: The frequently utilized Diode Pumped Solid State (DPSS) lasers emitting 532 nm (green) light can exhibit fluctuations in output power due to temperature and input power alterations. The polarization properties of the Nd:YAG and Nd:YVO4 crystals commonly used in these lasers are another potential source of fluctuation, since one means of controlling output power uses a polarizer with a variable transmission axis. Thus, the properties of DPSS lasers and the relationship between power output and infarct size were explored.

Results: DPSS laser beam intensity showed considerable variation. Either a polarizer or a variable neutral density filter allowed adjustment of a polarized laser beam to the desired intensity. When the beam was unpolarized, the experimenter was restricted to using a variable neutral density filter.

Comparison with existing method(s): Our refined approach includes continuous monitoring of DPSS laser intensity via beam sampling using a pellicle beamsplitter and photodiode sensor. This guarantees the desired beam intensity at the targeted brain area during stroke induction, with the intensity controlled either through a polarizer or variable neutral density filter.

Conclusions: Continuous monitoring and control of laser beam intensity is critical for ensuring consistent infarct size.

Keywords: Continuous monitoring of laser intensity; DPSS laser stability; Intensity adjustments; Laser beam polarization; Photothrombotic stroke; Variability.

Fig. 1

(A) Schematic of the laser system. L—lens; IA—intensity adjustment module. Note: a power meter is mounted beside the pellicle beamsplitter to monitor the sampled beam. (B) Photograph of an assembled laser system with a variable neutral density (ND) filter mounted as an “intensity adjustment” (IA) element. ND cage—the cage for a ND filter; D. Mirror—dielectric mirror; RP cage—the cage for a rotational polarizer (RP).

Fig. 2

(A) Output beam power P₁ as a function of the polarizer angle (θ_p). C—conversion factor; P₁—a fraction of the beam targeting the brain during stroke induction. (B) P₁/P₂ ratio as a function of θ_p. P₂—a fraction of the beam (split into fractions by a beamsplitter) continuously monitored by a sensor during the stroke induction.

Fig. 3

Representative images of a dorsal view of the rat brain (top panel) and a coronal brain section at 0.7 mm anterior from bregma (bottom panel) after photothrombotic stroke to the caudal area of the forelimb motor cortex. Cortical infarcts were induced by transcranial illumination with a green laser (532 nm) with a power density of 280 mW/cm² in combination with 10 mg/kg of Rose Bengal. The high variability in infarct size among rats is illustrated by (A) an infarct of the desired size, (B) a smaller than desired infarct and (C) no signs of infarct.

Fig. 4

Temporal profile of output laser power. (A) The beam was generated by a Diode Pumped Solid State (DPSS) laser without stabilization circuitry. Note the spontaneous drop from 103 mW (desired operation point) to 85 mW followed by recovery. (B) The beam was generated by a DPSS laser with stabilization circuitry. Note intensity stability throughout the experiment.

Fig. 5

(A) Output beam power as a function of polarizer angle (θ_p). P₁—a fraction of the beam targeting the brain during stroke induction. P₂—a fraction of the beam (split into fractions by a beamsplitter) continuously monitored by a power meter during the stroke induction. (B) Output beam intensity (measured at the skull) as a function of polarizer angle (θ_p). The beam is 4 mm in diameter at the skull surface.

Fig. 6

Representative images of a dorsal view of the rat brain (top panel) and a coronal section at 0.7 mm anterior from bregma (bottom panel) after a photothrombotic stroke to the caudal area of the forelimb motor cortex. (A) 280 mW/cm² beam and (B) 300 mW/cm² targeted the skull and underlying brain tissue. A higher beam intensity induced a bigger infarction.

Fig. 7

Output beam power as a function of a variable neutral density (ND) filter angle (θ_ND). P₁—a fraction of the beam targeting the brain during the stroke induction. P₂—a fraction of the beam (split into fractions by a beamsplitter) continuously monitored by a sensor during the stroke induction.