Cortical cooling refers to the cooling methods restricted to the cerebral cortex, where most higher brain processes occur. Neuroscientists generate various studies to help explain many of the complex connections and functions of the brain. Most studies utilize animal models that have varying degrees of comparison to the human brain; for example, small rodents are less comparable than non-human primates. One of the most definitive ways of determining which sections of the brain contribute to certain behavior or function is to deactivate a section of the brain and observe what behavior is altered. Lowering the temperature of the neural tissue has the effect of reducing metabolic activity in the affected region. This metabolic suppression slows down or inhibits neural firing, synaptic transmission, and other cellular processes that rely on energy. Below is a list of current cooling methods, their advantages and limitations, and some studies that have used cooling to elucidate neural functions.
Methods of neural tissue cooling There are a few options for cooling neural tissue; the chosen method depends on the experimental design including the section of the brain being cooled (the section of interest) and the volume of that section.
Cryoloops Cryoloops are cooling devices that use 23 gauge stainless steel hypodermic tubing shaped into a loop that can fit into sulci or on the gyri of the section of interest of the cerebral cortex. A pump draws methanol from a reservoir, and the fluid flows through a dry ice bath to be cooled. The cooled methanol flows through Teflon tubing into the cryoloop’s metal tubing, which is secured by passing through a threaded post. A thermocouple connector receives the wires from a microthermocouple at the base of the loop (where the inflow and outflow tubes meet) that measures the tubing temperature. The post, the thermocouple connector, and heat-shrink Teflon tubing, which envelopes the microthermocouple wires and the inflow and outflow tubes between the post and microthermocouple, are sealed using dental acrylic. After implantation, when the animal is not participating in an experiment, a protective cap is placed over the open inflow and outflow tubes. During experimentation, the inflow and outflow tubes are attached to the Teflon tubing connected to the reservoir setup. The thermocouple connector is connected to a switchbox and a thermometer so cryoloop temperature can be monitored.Lomber, S.G. (1999). The advantages and limitations of permanent or reversible deactivation techniques in the assessment of neural function. Journal of Neuroscience Methods, 86, 109-117.Lomber, S. G., Payne, B. R., & Horel, J. A. (1999). The cryoloop: an adaptable reversible cooling deactivation method for behavioral or electrophysiological assessment of neural function. Cryoloops are considered the most adaptable form of cooling available due to the customization necessary for each experiment. The investigator must form the functional cooling loop of the cryoloop to fit with whatever part of the brain he/she wishes to study, and several cryoloops can be used for one brain. Each device can cool a range of tissue areas from less than 10 mm3 to 75 mm3. Although it may be considered a hassle to have to form each device for each section of interest, this customization allows for a more controlled region of cooling and for more efficient animal use because of the possibility for multiple cooling sites within each animal. Head restraint is unnecessary because the loops are chronically implanted and secured to the skull by screws and dental acrylic.
Cooling plates Cooling plates are flat devices that are generally circular in shape and can cool tissue volumes of 35mm3 to 100mm3, usually by using thermoelectric cooling. Some investigators may use a similar setup to one necessary for the cryoloop to cool the plate (flow of coolant through a dry ice bath).Staba, R. J., Brett-Green, B., Paulsen, M., & Barth, D. S. (2003). Effects of ventrobasal lesion and cortical cooling on fast oscillations (> 200 Hz) in rat somatosensory cortex. [Article]. Journal of Neurophysiology, 89(5), 2380-2388. However, the electrical connections required for cooling are a simpler method than the setup necessary for tubes filled with coolant. To ensure the stability of the plate upon implantation, the animal must undergo fixed-head restraint, which limits the type of behavior that can be studied. Also, plates cannot conform to some areas of the cerebrum due to the disparate shapes of the plate and the brain, and they have not been successfully introduced into sulci.
Cryotips Cryotips are made of two stainless steel hypodermic-needle tubes, such as 18 gauge tube surrounding a 24 gauge tube, soldered together. Like the cryoloop, cooled methanol flows through the inner tube to cool the device. If the investigator chooses to insulate the shaft of the tube, a low-resistance heater-wire can be wrapped around the outer tube except for 2 mm at the tip; passing a direct current through the wire keeps the shaft at normal brain temperature. This ensures localized cooling at the tip, which is inserted into the brain to reach deeper structures, without cooling the overlying structures. More than one microthermocouple is required to measure shaft and tip temperatures.Skinner, J. E., & Lindsley, D. B. (1968). Reversible cryogenic blockade of unrestrained animals. [Article]. Science, 161(3841), 595-597. Modified versions of this device use smaller gauge tubing (21 and 30 gauge), and additional tubing is attached to form a y-shaped fork with HFC-134a cooling agent flowing through the double tubing while the fork is under vacuum. The vacuum causes the coolant to flow from the inner tubing into the outer tubing (so the coolant is in between the inner and outer tubes as well as inside the inner tube).Wang, Y., & Chambers, K. C. (2001). The role of the dura in conditioned taste avoidance induced by cooling the area postrema of male rats. [Review]. Behavioural Brain Research, 122(2), 113-129. Cryotips are usually used to cool deeper structures of the brain that cannot be thermodynamically cooled from the surface. They are not used much in cortical cooling due to the small volumes that are cooled – investigators cooling cortical tissue are usually interested in larger sections than this device can cool. Cryotips cool tissue volumes of 2 mm3 to 5 mm3. Usually the shaft of the device is insulated or even heated for localized cooling, however, some studies have used uninsulated cryotips to cool surface structures in addition to the deeper sections.Chambers, K. C., & Wang, Y. (2006). Cortical cooling induces conditioned consumption reduction in male rats. [Article]. Behavioural Brain Research, 172(1), 14-23.
Other Epileptic patients can undergo surgical resection to reduce the occurrence of seizures, and cortical stimulation mapping identifies functional neural tissue to preserve it. However, up to 5% of these patients will suffer from an intraoperative seizures during mapping. Recently, cooled saline was used during surgical resection in some of these patients and was found to reduce intraoperative epileptiform discharges (electroencephalogram spike frequency decreased), suggesting the potential of intraoperative seizure can be decreased by cooling the tissue.Ablah, E., Tran, M. P., Isaac, M., Kaufman, D. A. S., Moufarrij, N., & Liow, K. (2009). Effect of cortical cooling on interictal epileptiform activities. [Article]. Seizure-European Journal of Epilepsy, 18(1), 61-63.
Advantages and limitations A common method of deactivation when studying brain function is ablation of neural tissue, but there are several drawbacks. The exact location and extent of ablation, whether caused by chemicals or lesions, can only be defined post mortem. If the ablation occurred in an undesired location or has deactivated more of the tissue than intended, the time and resources were already been spent while obtaining results unrelated to the designed investigation. Also, ablation permanently deactivates the section of interest due to damage or removal of the neural tissue. Since the tissue cannot be reactivated, control measures that can be directly compared to the deactivation-induced effects cannot be obtained. Comparisons must be made between animals, which will have inherent differences, so internal double dissociations are not possible. Another major drawback in using ablation to deactivate tissue is that because the brain is plastic, while animals are recovering from ablation surgery, the cerebral cortex is able to modify the neural networking by activating new connections or strengthening pre-existing ones. This could cause the resulting behavior in the investigation to appear normal even though part of the animal’s brain has been deactivated, and then investigators would not be able to tell the contribution of the deactivated section to normal function. To overcome many of these drawbacks, cortical cooling devices may be used instead of ablation. While allowing a range of tissue areas to be cooled (small with cryotips to very large if using multiple cryoloops or a cooling plate), using cooling devices is a reversible method that enables control of the period of inactivation and, when turned off, takes only minutes for the animal to recover full function. These advantages hold even when deactivations are repeated over long periods of time, from months to years, with no evidence of attenuation.
Absence of neural compensation Cortical cooling devices do not cause any damage to the neural tissue when they are implanted or used repeatedly to cool the section of interest. This allows reversal of the deactivation and eliminates the concern of neural compensation. Cooling can be quickly initialized and terminated with the currently available devices, so the neural tissue has no time to create or strengthen neural networks. This ensures the deactivation induces an effect in neural function, and the behavior being studied is produced from the deactivated tissue and not from modified networks.
Efficient animal use Reversibility of the deactivation enables animals to be used as their own controls, which removes variation between animals designated as “control” and animals in the experimental group and allows for internal double dissociations. Large amounts of data can be gathered for each animal since it can undergo several trials in one experiment or, in the case of the chronically implanted cryoloops and cryotips, be used in more than one experiment. These advantages allow for fewer animals needed for each experiment while obtaining reliable results.
Control of deactivated tissue parameters Based on thermodynamic principles, thermoclines can be determined to establish the spread of cooling from particular cooling surfaces. Therefore, for each cooling device with a known and consistent surface area, the temperature can be set at the same value for each trial or experiment to generate the same thermoclines and replicate the same volume of deactivation. Therefore, specifically selected regions of tissue can be reversibly deactivated in a controlled and reproducible way. 20°C has been found to be the critical temperature for active neuronal signals; below this temperature, afferent signals cannot activate neurons and the tissue is considered deactivated. As long as the desired tissue reaches below the critical temperature while the surrounding tissue remains above it, the thermoclines generated by the device can be pre-calculated so that the temperature can be set to only deactivate the tissue of interest. Cooling can also be initiated and terminated with the same time required to reach either deactivation temperature or normal physiological temperature each time. This enables control over the onset of deactivation, its duration, and recovery for each experiment.
Experimental limitations due to physical setup