By Barrett St. George, Diane Cheek, Alyssa Everett & Frank E. Musiek, PhD
The University of Arizona, Department of Speech, Language and Hearing Sciences
Occasionally there is a breakthrough in science, basic or clinical, that affords investigators a definite advantage in their quest for more knowledge and solutions. Sometimes the uptake of this new approach takes a period of time before its value is fully realized. The cryoloop cooling technique is one to which these opening comments apply.
Cryoloop cooling can create a lesion in the central nervous system by deactivating neural responses, but then it can be reversed. This allows measurements before, during and after applying the technique. This can allow more accurate measurement of effects and a return to baseline function. It helps preserve the test animal’s functional integrity and provides insights to disordered systems that have not been achieved before.
Lesion effects are key to understanding disordered systems, so the use of cryoloop cooling should lead to improvements in diagnostic and rehabilitative audiology. Therefore we have decided to provide a glimpse into this technique and how it works. Like many advances, it is not totally new, but is fast attracting interest in many areas of neuroscience.
Purpose of Cortical Cooling and its History
When cortical neurons are cooled to a particular temperature, their synaptic activity halts until the cooling stops and they can resume pre-cooled synaptic function (Lomber, Payne, & Horel, 1999). This reversible neural deactivation technique enables researchers to investigate cortical organization and its effect on behavior without causing the permanent cortical damage that accompanies traditional ablation techniques.
Cortical cooling techniques examining the neural contributions of behavior have been in existence for over 100 years (Brooks, 1983). However, in the last few years these methods have become the subject of considerable interest in the research community. In an effort to improve accuracy and efficacy, the sophistication of cortical cooling techniques has evolved substantially.
Brooks (1983) and Lomber (1999) examined this evolution process, which began in 1910 with direct brainstem and cortex cooling in an effort to examine autonomic reflexes and motor movement in animals. In the 1970s, the thermoelectric plate was developed, which could more precisely control the cooling process by incorporating the Peltier effect into the cooling technology.
Briefly, the Peltier effect cools thermoelectric plates by passing an electric current between two conductor plates within the device. This electric current creates a heat transfer between the plates where heat is absorbed by one plate (on the cortex) and is transferred to and dissipated by the other plate (Active Cool, n.d.). The thermoelectric plates’ ability to cool relatively large cortical surface areas quickly made it an attractive cooling device among researchers.
While thermoelectric plate cooling can be used to examine behavioral deficits, the extent to which behavior can be examined is limited by the requirement of having the test animals’ heads restrained. Additionally, the physical construction of the thermoelectric plates restricts surface cooling to areas no less than 35 mm3 and denies researchers the ability to custom-shape the cooling surface to conform to cortical surfaces.
Cryotips, developed in the 1960s and technologically refined since then, answered the need for precise subcortical deactivation (Skinner & Lindsley, 1968). These needle-like devices penetrate through the cortical surface to deliver precise cooling via the device’s tip. Lomber (1999) noted that, unlike chemical deactivation, which is also used to deactivate subcortical regions, cryotips give researchers more deactivation control without the inherent risk of chemical spread into undesired cortical areas. This technique is often limited to small, focal subcortical areas, as irreversible damage to the cortical surface occurs as a result of the cryotip’s cooling delivery method.
Not until the early 1980s was an effective and highly adaptable cooling device developed for cortical research. This device, the cryoloop, was first used in visual discrimination research in cryoloop-implanted monkeys (Horel, Voytko, & Salsbury, 1984). Since then, cryoloops have been successfully used in cats, guinea pigs, and rats, in both acute and chronic applications, to map the functional organization and reorganization of auditory, visual, motor, and parietal cortices (Brown & Teskey, 2014; Cooke et al., 2012; Lomber, Meredith, & Kral, 2010)
Cryoloop Construction and Operation
The cryoloop device, as described by Lomber et al. (1999), is relatively inexpensive to construct (Figure 1). Using a brain replica and simple hand tools, 23-gauge stainless steel tubing is shaped into a loop customized to the desired cortical area to be deactivated. Individual loop surface areas range from 10 to 100 mm3, but may be clustered together if larger areas need to be deactivated.
A microthermocouple wire (copper and constantan wire) is soldered to the loop and monitors the loop temperature during cooling procedures. The microthermocouple wire travels from the loop through clear, protective tubing, into a thermocouple connector (female adaptor jack), which connects to an external thermometer. Dental acrylic fastens this connector to the cryoloop device. The two open ends of the cryoloop tubing serve as inlet and outlet tubes through which cooled methanol flows. These tubes also travel through the clear tubing and are housed inside a stainless steel cylinder adjacent to the connector.
When not in use, the inlet and outlet tubes are protected by a stainless steel cap that screws onto the cylinder housing. During cooling procedures, this protective cap is removed and Teflon tubing is attached to the exposed inlet and outlet tubes.
So, how is this cryoloop device cooled and how is that temperature maintained? The cryoloop is cooled by an external cooling system (Lomber et al., 1999) (Figure 2). Beginning at a methanol reservoir, a pump drives methanol from the reservoir through Teflon tubing, which is partially submersed in a methanol/dry ice bath. Once in this ice bath, the methanol within the tubing can reach temperatures as cold as -75°C.
After it is chilled, the methanol is pumped through the inlet tube to the cryoloop, where it will cool the desired cortical area. The methanol then exits the cryoloop via the outlet tube and is deposited back into the methanol reservoir, where it will cycle through the cooling system once again. In experiments with cats, harnesses worn by the animal manage the microthermocouple wire and Teflon tubing to prevent impedance on the behavioral task (Lomber & Payne, 2001). Separate cooling systems can be set up to independently control or sequence multiple cryoloops implanted in a single animal (Salsbury & Horel, 1983). Long-term maintenance of the external cooling system involves keeping the methanol reserve clean and checking the Teflon tube for leaks (Lomber et al., 1999).
Proper cryoloop temperature is maintained by controlling the distance between the ice bath and cryoloop, which Lomber et al. (1999) recommend be no greater than three feet. Other factors influencing cryoloop temperature include the methanol’s pump rate and the brain’s internal temperature. However, as Cooke et al. (2012) point out, cortical temperature monitoring during behavioral tasks has not yet been accomplished in chronically implanted animals.
In other words, during behavioral tasks, researchers monitor the cryoloop’s temperature rather than the temperature of the surrounding cortical tissue. Therefore, cortical temperature maps obtained from electrophysiological measures during acute cryoloop preparations help researchers maintain proper cryoloop temperature during behavioral tasks. Maintaining proper cryoloop temperature also requires knowledge of microthermocouple timing, as there is a two- to three-minute delay between the temperature measured by the microthermocouple and the actual cortical temperature (Lomber et al., 1999).
Cryoloop Implantation and Viability
Cryoloops can be used in acute preparations during craniotomy in anesthetized animals or they may be surgically implanted for long-term behavioral tasks (Lomber, 1999). As Lomber et al. explain, the total number of cryoloops that can be implanted in a single animal is restricted by the animal’s brain size and the length of surgery. The cat cortex has a six-cryoloop maximum capacity, bilaterally, which would require a 10-hour implantation surgery. The monkey cortex has a ten-cryoloop maximum capacity, bilaterally, which would inherently require a longer surgical procedure.
The surgical implantation procedure begins with a craniotomy over the desired cortical area, with the animal under general anesthesia. A sterilized cryoloop is then positioned on the cortical surface or within the sulci. The cryoloop device is fastened to the skull using dental acrylic on bone-anchored screws. Finally, the skull and scalp are surgically repaired and the animal receives post-operative care, as required. Once the animal has recuperated, periodic antiseptic care is provided to prevent infection around the device.
Long-term viability of implanted cryoloops is species dependent. In cats, cryoloops have been successfully implanted for up to three years, while in monkeys, infection and skull deterioration are not uncommon within the first year of implantation (Lomber et al., 1999).
Cryoloop Clinical Relevance
Delivery of reversible cortical deactivation in a relatively straightforward but highly versatile design has made cryoloop cooling an attractive tool in many areas of cortical research, as previously mentioned. Within the realm of auditory research, cryoloop cooling has recently been utilized to behaviorally confirm the characteristic responses of neurons in particular auditory cortex regions. This technique therefore aids in the determination of the functions of different cerebral areas and pathways.
A major area of auditory research has been the investigation of sound localization due to the implications of its clinical significance. Lomber and Malhotra (2008) found that sound-localization deficits were generated in the cat by bilateral deactivation of posterior auditory regions; whereas pattern-discrimination deficits were created by bilateral deactivation of anterior auditory regions and not vice versa. This double disassociation suggests that the information processed in the auditory cortex is segregated into regions with different processing streams likely inherent.
Investigating sound localization and pattern discrimination abilities in stroke patients may confirm the results of this study in humans, as it is well known that pattern perception tests are used in the clinical evaluation of individuals with central auditory system dysfunction. Based on the information garnered from this 2008 article, it would not be surprising if individuals with middle cerebral artery (MCA) lesions affecting anterior auditory regions demonstrate pattern-discrimination deficits. Similarly, it can be expected that MCA lesions affecting posterior auditory regions produce sound localization deficits.
A major study by Malhotra, Hall, and Lomber (2004) examined sound localization abilities of cats during unilateral cooling of 19 specific cerebral areas. This study, in particular, is a great example of how flexible the cryoloop deactivation method is for investigating multiple lesion sites in the same animal. Up to six cryoloops were implanted over multiple cerebral loci in each cat.
This study demonstrated that, in addition to a healthy primary auditory cortex, the integrity of other auditory cortical areas is vital to the cat’s ability to localize accurately. It appears that the cat’s posterior auditory field and the anterior ectosylvian sulcus need to be intact for accurate sound localization. By using the cryoloop procedure, this study provides additional and specific information on a well-known phenomenon seen clinically in that people with auditory cortex lesions suffer in their ability to localize sound.
The functionality of auditory cortex regions involved in sound localization has also been scrutinized in a three-dimensional manner. The cyroloop cooling technique has allowed for this investigation of cortex function in the third dimension, i.e., depth. As discussed previously, unlike traditional lesioning techniques, cryoloop cooling allows for exceptionally focal deactivation of cortical areas.
Using a method of graded cooling, Lomber, Malhotra and Hall (2007) were able to show that laminar contributions to sound localization exist in the auditory cortex. They found that deactivating only the superficial layers of certain auditory areas created significant sound localization deficits. This research suggests that sound localization deficits are associated with the depth of auditory cortex injuries. Thus, certain undercut lesions that occur deeper within the cortex may have no effect on acoustic orienting. On the other hand, superficial lesions are likely to produce profound deficits of sound localization. This new insight on the anatomically-related relevance of central auditory areas involved in sound localization holds significant clinical value in understanding the implications of location-specific damage to the auditory cortex.
Cryoloop deactivation of the auditory cortex has also been shown to affect the neuronal responsiveness to interaural level differences at the level of the inferior colliculus (Nakamoto, Jones & Palmer, 2008). This effect has significant contributions to the ability to localize as well, especially for high frequencies evidenced by the head shadow effect.
It has long been unclear exactly how the inferior colliculus contributes to auditory scene analysis. Cryoloop studies of auditory cortex have shed further light on the functions of the upper efferent system pathways influencing this structure.
For example Nakamoto, Shackleton and Palmer (2010) found that global cooling of auditory cortex has a specific influence on the synchronicity of neurons in the inferior colliculus; the effect is a decrease in the difference of neural synchronization to concurrent harmonic stimuli as well as a decrease in overall response magnitude. In other words, healthy descending pathways of the auditory cortex play a major role in the spectral discrimination of sounds by modulating the way neurons in the brainstem respond to auditory stimuli.
The information garnered from this study is clinically relevant for individuals with lesions to the auditory cortex or the descending auditory pathway. These individuals have shown difficulties perceptually segregating sounds in noisy environments, which may, in fact, be partially due to the lack of top-down information received at the brainstem from the auditory cortex. This type of knowledge concerning the neuroanatomical areas that contribute to sound localization ability is needed for a comprehensive understanding of the clinical significance of lesions to particular areas of the central auditory system.
Cryoloop cooling has enhanced our knowledge of the descending auditory pathway in that studies have shown that cooling of upper/cortical areas has effected responses as far caudally as the cochlea by elecrocochleographic recordings. A recent cryoloop study investigating this cortical descending control demonstrated that global cooling of the auditory cortex resulted in decreased amplitudes of the cochlear microphonic and compound action potential in anesthetized guinea pigs, suggesting that the auditory cortex exercises control over the physiological processes in the cochlea and auditory nerve even when the animal is comatose (Edwards & Palmer, 2010).
Deactivation of auditory cortex basal activity was found to modulate the amplitude of cochlear and auditory nerve responses in anesthetized chinchillas as well (Leon, Elgueda, Silva, Hamame & Delano, 2012). Future studies using more precise spatial deactivation of auditory cortex areas are essential for a better understanding of the descending pathways influencing the peripheral auditory system. Focal auditory cortex deactivation of regions that modulate the olivocochlear system could be a way to suppress extraneous neural activity, therefore providing relief for certain individuals with tinnitus.
The unilateral deactivation of particular auditory cortex areas generates profound localization deficits to acoustic stimuli in the contralateral hemifield (Sanchez-Longo & Forster, 1956).
This is a major issue for individuals with lesions in the auditory cortex. A recent animal model cryoloop study may have discovered how to remediate the sound localization deficits engendered by such lesions. Lomber, Malhotra and Sprague (2007) have found that deactivation of the superior colliculus contralateral to the impaired hemifield restores sound localization deficits caused by auditory cortex lesioning. It is therefore possible that surgical restoration of acoustic orienting will soon become an option for individuals with unilateral auditory cortex lesions. However, additional research replicating this effect is necessary before such radical clinical progress can be made. (Sanchez-Longo & Forster, 1958)
Evidence that Cryoloop Cooling Works
A few techniques have been used to provide evidence that cryoloop cooling works as measured scientifically. The preponderance of research utilizing the cryoloop deactivation method combines neurophysiological and thermal measures to verify and quantify the effectiveness of cooling. Single-unit electrophysiological recordings at specific cortical locations verify the neuronal responses near the cryoloop before, during and after cooling. This provides precise information regarding the effect of cooling on neuron activity. Almost immediately, cooling produces extremely reduced activity of neurons, which return to normal after the cryoloop is turned off. Neurons are equally responsive before and after cooling, suggesting that the cooling does not harm cortex. Post-mortem histological procedures have verified this as well, presenting no visual sign of damage.
In addition to neurophysiological methods, thermal measures also provide important information about the effect of cooling on the brain. Small thermometers, called microthermocouples, are used to record temperature across the cortex. This, in turn, provides information about the extent of deactivated cortex induced by cryoloop cooling.
Neurophysiological and thermal measures have been used in conjunction to determine the temperature at which neurons fail to be activated by afferent signals (20°C). These two techniques have also been used together to examine the spread of cooling from the cryoloop. Even when the cryoloop temperature is reduced to 1 degree Celsius, the effect of cooling is confined to only 1.5 -2.5 mm distance from the cryoloop (Lomber et al., 1999). The microthermocouple measurements have accurate temporal tenacity, and can therefore provide information about the rate of spread of cooling throughout the cortex.
Knowledge of arterial vasculature is important prior to implantation of cryoloop because cooling effects in cortex are spread actively by chilled blood and not passively. Temperature measurements often reveal asymmetrical cooling effects from cryoloop across cortical tissue due to differences in the underlying arterial vasculature (Lomber et al., 1999). Both neurophysiological and thermal measuring techniques during cryoloop cooling are completed to confirm the accuracy of what is being measured.
Advantages and Disadvantages
Cryoloop cooling is an adaptable procedure that surpasses the previously utilized techniques to assess neural function as an effect of creating lesions. Like all procedures, it has drawbacks; however, cryoloop cooling allows the cerebral cortex to be analyzed without irreversible implications. Specifically, the cryoloop primarily cools the cortical surface affecting gray matter more than white matter. Malhotra et al. (2004) experimented with cats in 19 cortical sites and found after months of repeated deactivation that cryoloops did not influence the baseline functioning of these areas.
The materials used for cryoloop cooling and the viability of utilizing fewer animals make it maintainable at a modest cost. As stated previously, these materials also make the cryoloop easily adaptable to each subject’s brain shape and size, allowing selected regions of the cortex to be cooled independently of surrounding structures.
Suppressing brain function previously involved creating permanent lesions that typically induced neural compensations requiring head restraints and more animal subjects. Cryoloop cooling allows for repeated coolings on the same animals over long periods of time because it is reversible and produces stable, non-compromising conclusions. Fewer animals are needed to show reliable results because the animals tested can also be used as the control subject group when the cooling is reversed (Lomber, 1999).
When a lesion is made on the cortex, the animal will learn over time to compensate for this handicap and conclusions will be skewed due to this adaptation (Lomber, 1999). However, because cryoloop cooling is a relatively short process, the animal will not have time to adapt to the handicap. Cryoloops are capable of effectively cooling deep brain structures and can deactivate neurons in less than one minute. After the process is over, the neurons begin to reactivate at the same rate as deactivation (Lomber et al., 1999). Repeated testing does not result in compromised neural activity following the procedure (Lomber et al., 1999).
The implications for cryoloop cooling are far less than that of lesions, although there are drawbacks to this method as well. The purpose of using cryoloop cooling on animal subjects is to attempt to mimic the cortical damages that a human may encounter by looking at behavioral deficits. However, comparing human deficits to reversible animal deficits is difficult when the animal does not actually have a physical impairment (Lomber, 1999). The cryoloop itself, although removable, can leave a small impression on the cortical surface in which repeated use over a short period of time may affect neural functioning to a minor degree (Lomber, 1999). Also, as with any surgical procedure, there are always risks of human error. The cryoloop needs to be secured to the exact location or cooling may not be as effective.
When a lesion is made on the cortex, those neurons and synaptic clefts are destroyed, whereas cooling temporarily disables them. However, afferent axons and a small number of neurons will still remain active during cooling leaving room for some doubt in the interpretations (Lomber et al., 1999). Cooling the cortex may inhibit the brain function in that area, but it also cools the blood that circulates away from the specific area. The cooled blood can spread 1.5 mm laterally and 2.5 mm medially, which can affect and temporarily disable those regions as well (Lomber et al., 1999). These effects in the cortex from the cooled blood may lead to misinterpretations of neural dysfunction in the target area.
Neuron activation and deactivation may only take about a minute, but the effects on behavior do not recover as quickly. It takes about two to three minutes after the cooling has stopped for body temperatures affected by the process to return to normal (Lomber et al., 1999). Behaviorally, reversal of impairments may take as long as or longer than it would take for body temperatures returning to normal. Cooling brain structures rather than creating a lesion in the traditional manner has a slightly reduced specificity of location due to the blood cooling and circulating.
Overall, the cryoloop’s modest cost, straightforward operation, and customizability has made it an attractive cooling device to create reversible neural deactivation in cortical research.
Barrett St. George is a first year Doctor of Audiology student and research assistant at the University of Arizona. He graduated magna cum laude from the University of Connecticut with a BA in Communication Disorders. He has worked for Frank Musiek, Ph.D. for close to three years, assisting in the UConn Neuroaudiology and Vestibular labs. His primary interests are in vestibular vascular anatomy and vestibular research pertaining to the aerospace environment. One of his ambitions is to utilize hyperbaric chambers to study vestibular function in both hypoxic and hyperoxic environments.
Diane Cheek is a first-year student in the Doctor of Audiology program at the University of Arizona. She graduated with honors from the University of West Florida with a BA in International Studies and later served eight years as an Intelligence analyst in the United States Air Force. Diane enjoys all aspects of diagnostic and rehabilitative audiology, particularly matters concerning auditory processing. She is currently involved in research investigating infant speech perception using cortical auditory evoked potentials. Additionally, Diane serves as the University of Arizona’s Student Academy of Audiology secretary and is actively involved with philanthropic events aimed at advancing the audiologic health of southern Arizona’s underserved community.
Alyssa Everett is a first year doctoral student of Audiology at the University of Arizona, expected to graduate in May of 2018. She completed her undergraduate degree in Speech Pathology and Audiology at Towson University, graduating with honors. She has many interests in this field, especially research in electrophysiology and central auditory processing. For her Au.D. Capstone research project at U of A, Alyssa is examining acceptable noise levels in adults to better understand why people may or may not use their assisted listening devices. Interested in international studies and culture, Alyssa studied in Italy and the Dominican Republic to gain a deeper understanding of their culture.
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