Dieses Protokoll ermöglicht Axoplasma Isolation mit der Minimierung der Glia-und Gefäßgewebe Kontamination. Die Methode liefert etwa 70-100 ug Gesamt Axoplasma Protein pro 8-10 Wochen alte Wistar-Ratte. 1. Dissect Ischiasnerven <ol><li> Euthanize zwei Ratten durch CO 2-Inhalation durch Genickbruch folgte. Bestätigen Tod durch Palpation wegen fehlender Herzschlag vor Beginn der Dissektion. </li><li> Swab der Präparation mit 70% Ethanol. </li><li> Schneiden Sie die Haut, separate Muskeln und isolieren Ischiasnerv mit einer Schere und Pinzette vorsichtig ohne Beschädigung der Blutgefäße. Dissect sowohl Ischiasnerv (ca. 1,5 cm) von jedem Tier und sammeln Sie alle vier Nerven in einer Eppendorf mit 500 ul PBS von 0,2 + Inhibitoren, auf Eis gehalten. </li><li> Übertragen Sie die Nerven Segmente Plastikschalen mit mindestens 2 mL PBS von 0,2 + Protease-Inhibitoren. </li><li> Entfernen Sie das Epineurium von den Nerven mit feinen Pinzetten unter dem Binokular Umfang. Trennen Sie die Faszikel sehr sorgfältig, bis sie bedeckt werden und beginnen, auf der Oberfläche der Lösung schwimmen. Dieser Schritt sollte geübt werden, bis sie schnell und präzise durchgeführt werden können (nicht an mehr als 10 Minuten pro Nerv) werden. </li></ol> 2. Inkubation, Waschen und Elution <ol><li> Übertragung getrennt Faszikeln in ein frisches Eppendorf-Röhrchen mit 500 ul PBS 0.2X mit Protease-Inhibitoren für die Inkubation für 2 Stunden bei Raumtemperatur. </li><li> Wash mindestens 3-mal mit 1 mL des gleichen Puffers durch die Übertragung der Faszikel von Eppendorf-Röhrchen zu Eppendorf-Röhrchen und Schütteln für 5 min nach dem Transfer. </li><li> So entfernen Sie überschüssige Flüssigkeit legte die Faszikel in eine neue, leere Eppendorf-Röhrchen und übertragen dann die Faszikel, um eine neue Eppendorf-Röhrchen mit 300 ul PBS 1X mit Protease-Inhibitor für die Inkubation für 20-30 min bei RT. </li><li> Zentrifuge 10 min bei 10.000 xg bei 4 ° C. </li><li> Nehmen Sie den Überstand und messen Proteinkonzentration. Dies ist Ihr Axoplasma Vorbereitung. </li></ol> 3. Repräsentative Ergebnisse <img src="/files/ftp_upload/2087/2087fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Western-Blot-Vergleich Axoplasma durch die manuelle Squeeze-Methode mit Axoplasma Proben isoliert, wie in diesem Protokoll isoliert dargestellt. Beachten Sie die reduzierte Mengen an Albumin und GFAP, was Blut Protein-und Gliazellen Verunreinigungen beziehungsweise. Im Gegensatz dazu ist axonalen Tubulin b3 in dieser Zubereitung angereichert, was auf eine hohe axonale Proteine.

<ol>
	<li>Hanz, S., Perlson, E., Willis, D., Zheng, J. Q., Massarwa, R., Huerta, J. J., Koltzenburg, M., Kohler, M., van-Minnen, J., Twiss, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axoplasmic+importins+enable+retrograde+injury+signaling+in+lesioned+nerve.">Axoplasmic importins enable retrograde injury signaling in lesioned nerve.</a> Neuron. 40, 1095-1104 (2003).</li><li>Michaelevski, I., Medzihradszky, K. F., Lynn, A., Burlingame, A. L., Fainzilber, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+transport+proteomics+reveals+mobilization+of+translation+machinery+to+the+lesion+site+in+injured+sciatic+nerve.">Axonal transport proteomics reveals mobilization of translation machinery to the lesion site in injured sciatic nerve.</a> Mol Cell Proteomics. 9 (5), 976-987 (2010).</li><li>Rishal, I., Michaelevski, I., Rozenbaum, M., Shinder, V., Medzihradszky, K. F., Burlingame, A. L., Fainzilber, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axoplasm+isolation+from+peripheral+nerve.">Axoplasm isolation from peripheral nerve.</a> Dev Neurobiol. 70, 126-133 (2010).</li><li>Twiss, J. L., Fainzilber, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosomes+in+axons--scrounging+from+the+neighbors.">Ribosomes in axons--scrounging from the neighbors.</a> Trends Cell Biol. 19, 236-243 (2009).</li></ol>

Biochemische und Proteom-Analysen haben bestätigt, dass dieses Verfahren Serum und Gliazellen Verschmutzungsgrad 3 reduziert wie zuvor beschriebenen Verfahren zur Isolierung Axoplasma durch mechanische Squeeze 1 verglichen. Wir haben diese Axoplasma Isolation Protokoll verwendet, um Dynein-basierte retrograde Signalisierung nach Ischiasnerv Verletzung 2 zu erforschen, und erwarten, dass es Nutzen in der Erforschung von vielen anderen Prozessen in der Erwachsenenbildung peripheren Nerven...

<ul><li> Männliche Wistar-Ratten (8-10 Wochen alt) </li><li> PBS von 0,2 und PBS 1X Lösungen mit Protease-Inhibitoren (Rosche) 2 Tabletten pro 50 ml </li><li> Eppendorfs für 1,5 ml </li><li> Sterilisiert Plastikschalen 35 mm </li><li> Dissecting Tools wie: Schere und feinen Pinzette </li><li> Fernglas und Tischleuchte </li></ul> Antibodies Maus anti-GFAP Klon GA-5 wurde von Sigma (G6171). Kaninchen-anti-Albumin wurde aus Cedarlane (CLAG5140). Maus anti-Tubulin β3 und Kaninchen-Anti-Gerk wurden von Sigma (T2200 und M5670 jeweils).

axoplasm isolation from rat sciatic nerve

Isolierung von reinem axonalen Zytoplasma (Axoplasma) von peripheren Nerven ist entscheidend für biochemische Untersuchungen von vielen biologischen Prozessen. In diesem Artikel zeigen wir, und beschreiben Sie ein Protokoll für Axoplasma Isolierung von adulten Ratten Ischiasnerv auf den folgenden Schritten: (1) Zerlegung der Nervenfaszikel und Trennung von Bindegewebe, (2) Inkubation von kurzen Segmenten Nervenfaszikel in hypotonischen Medium Myelin Freigabe und lysieren nicht axonalen Strukturen, und (3) Extraktion der verbleibenden Axon-angereichertem Material. Proteomik und biochemische Charakterisierung dieser Zubereitung hat einen hohen Grad der Anreicherung für axonale Komponenten bestätigt.

Watch this Scientific Journal Video about Axoplasm Isolation from Rat Sciatic Nerve at JoVE.com

Axoplasm Isolation from Rat Sciatic Nerve

Wir zeigen, ein Protokoll für Axoplasma Isolierung von adulten Ratten Ischiasnerv auf Dissektion Nervenfaszikel und Inkubation in hypotonischen Medium Myelin Freigabe und lysieren nicht axonaler Strukturen, die durch Extraktion der restlichen Axon-angereichertem Material gefolgt basiert.

Axoplasma Isolation von Rat Ischiasnerv

Isolation of pure axonal cytoplasm (axoplasm) from peripheral nerve is crucial for biochemical studies of many biological processes. In this article, we ...

axoplasm-isolation-from-rat-sciatic-nerve

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

13.6K Aufrufe.  Weizmann Institute of Science. Wir zeigen, ein Protokoll für Axoplasma Isolierung von adulten Ratten Ischiasnerv auf Dissektion Nervenfaszikel und Inkubation in hypotonischen Medium Myelin Freigabe und lysieren nicht axonaler Strukturen, die durch Extraktion der restlichen Axon-angereichertem Material gefolgt basiert.

Serie: Axoplasm Isolation from Rat Sciatic Nerve

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

Reproduzierbare Maus Ischiasnerv Crush und anschließende Bewertung der Regeneration von Whole mount Muskelanalyse

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Forschung

Neurowissenschaften

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

Isolierung und Kultivierung von embryonalen Ratten-Nervenzellen: ein Quick-Protokoll

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological techniques, in the understanding of the underlying pathophysiology1. Here we describe a method to perform electrophysiological studies on mouse sciatic nerves in vivo.
The animals are anesthetized with isoflurane in order to ensure analgesia for the tested mice and undisturbed working environment during the measurements that take about 30 min/animal. A constant body temperature of 37 &#176;C is maintained by a heating plate and continuously measured by a rectal thermo probe2. Additionally, an electrocardiogram (ECG) is routinely recorded during the measurements in order to continuously monitor the physiological state of the investigated animals.
Electrophysiological recordings are performed on the sciatic nerve, the largest nerve of the peripheral nervous system (PNS), supplying the mouse hind limb with both motoric and sensory fiber tracts. In our protocol, sciatic nerves remain in situ and therefore do not have to be extracted or exposed, allowing measurements without any adverse nerve irritations along with actual recordings. Using appropriate needle electrodes3 we perform both proximal and distal nerve stimulations, registering the transmitted potentials with sensing electrodes at gastrocnemius muscles. After data processing, reliable and highly consistent values for the nerve conduction velocity (NCV) and the compound motor action potential (CMAP), the key parameters for quantification of gross peripheral nerve functioning, can be achieved.

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Measurements of nerve conduction properties in vivo exemplify a powerful tool to characterize various animal models of neuromuscular diseases. Here, we present an easy and reliable protocol by which electrophysiological analysis on sciatic nerves of anesthetized mice can be performed.

In-vivo-Elektrophysiologische Messungen an Maus Ischiasnerven

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

Minimally-invasive Technique for Injection into Rat Optic Nerve

The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central nervous system in a minimally-invasive surgery without bone removal. This technique describes an approach to visualization and direct injection of the optic nerve following minor fascial dissection from the orbital ridge, using a conjunctival traction suture to gently pull the eye down and out. Representative examples of an injected optic nerve show successful injection of dyed beads.

Direct injection into the rat optic nerve is useful for regenerative research. We demonstrate a minimally-invasive technique for direct injection into a rat optic nerve that does not involve opening the skull. Using this method, surgical complications are minimized and recovery is more rapid.

Minimal-invasive Technik für die Injektion in Ratten-Optic Nerve

The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central ...

Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to monitor the disease course in the follow-up period. Changes in the VEP parameters closely correlate with pathological damage in the optic nerve. This protocol provides a detailed description about the rodent model of optic nerve microinjection, in which a partial demyelination lesion is produced in the optic nerve. VEP recording techniques are also discussed. Using skull implanted electrodes, we are able to acquire reproducible intra-session and between-session VEP traces. VEPs can be recorded on individual animals over a period of time to assess the functional changes in the optic nerve longitudinally. The optic nerve demyelination model, in conjunction with the VEP recording protocol, provides a tool to investigate the disease processes associated with demyelination and remyelination, and can potentially be employed to evaluate the effects of new remyelinating drugs or neuroprotective therapies.

Focal demyelination is induced in the optic nerve using lysolecithin microinjection. Visual evoked potentials are recorded via skull electrodes implanted over the visual cortex to examine the signal conduction along the visual pathway in vivo. This protocol details the surgical procedures underlying electrode implantation and optic nerve microinjection.

Visuell evozierten Potential-Aufnahme in einem Rattenmodell für Experimentelle Optic Nerve Demyelinisierung

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

In vivo - Gentransfer auf Schwann - Zellen in der Nagerischiasnerv durch Elektroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing

Electrophysiology enables the objective assessment of peripheral nerve function in vivo. Traditional nerve conduction measures such as amplitude and latency detect chronic axon loss and demyelination, respectively. Axonal excitability techniques &#34;by threshold tracking&#34; expand upon these measures by providing information regarding the activity of ion channels, pumps and exchangers that relate to acute function and may precede degenerative events. As such, the use of axonal excitability in animal models of neurological disorders may provide a useful in vivo measure to assess novel therapeutic interventions. Here we describe an experimental setup for multiple measures of motor axonal excitability techniques in the rat ulnar nerve.
The animals are anesthetized with isoflurane and carefully monitored to ensure constant and adequate depth of anesthesia. Body temperature, respiration rate, heart rate and saturation of oxygen in the blood are continuously monitored. Axonal excitability studies are performed using percutaneous stimulation of the ulnar nerve and recording from the hypothenar muscles of the forelimb paw. With correct electrode placement, a clear compound muscle action potential that increases in amplitude with increasing stimulus intensity is recorded. An automated program is then utilized to deliver a series of electrical pulses which generate 5 specific excitability measures in the following sequence: stimulus response behavior, strength duration time constant, threshold electrotonus, current-threshold relationship and the recovery cycle.
Data presented here indicate that these measures are repeatable and show similarity between left and right ulnar nerves when assessed on the same day. A limitation of these techniques in this setting is the effect of dose and time under anesthesia. Careful monitoring and recording of these variables should be undertaken for consideration at the time of analysis.

In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing

Axonal excitability techniques provide a powerful tool to examine pathophysiology and biophysical changes that precede irreversible degenerative events. This manuscript demonstrates the use of these techniques on the ulnar nerve of anesthetized rats.

In Vivo Elektrophysiologische Messungen der Ratte ulnaris mit axonalen Erregbarkeit testen

Electrophysiology enables the objective assessment of peripheral nerve function in vivo. Traditional nerve conduction measures such as amplitude and ...

Facial Nerve Surgery in the Rat Model to Study Axonal Inhibition and Regeneration

This protocol describes consistent and reproducible methods to study axonal regeneration and inhibition in a rat facial nerve injury model. The facial nerve can be manipulated along its entire length, from its intracranial segment to its extratemporal course. There are three primary types of nerve injury used for the experimental study of regenerative properties: nerve crush, transection, and nerve gap. The range of possible interventions is vast, including surgical manipulation of the nerve, delivery of neuroactive reagents or cells, and either central or end-organ manipulations. Advantages of this model for studying nerve regeneration include simplicity, reproducibility, interspecies consistency, reliable survival rates of the rat, and an increased anatomic size relative to murine models. Its limitations involve a more limited genetic manipulation versus the mouse model and the superlative regenerative capability of the rat, such that the facial nerve scientist must carefully assess time points for recovery and whether to translate results to higher animals and human studies. The rat model for facial nerve injury allows for functional, electrophysiological, and histomorphometric parameters for the interpretation and comparison of nerve regeneration. It thereby boasts tremendous potential toward furthering the understanding and treatment of the devastating consequences of facial nerve injury in human patients.

This protocol describes a reproducible approach to facial nerve surgery in the rat model, including descriptions of various inducible patterns of injury.

Gesichtsnervenchirurgie im Rattenmodell zur Untersuchung der axonalen Hemmung und Regeneration

This protocol describes consistent and reproducible methods to study axonal regeneration and inhibition in a rat facial nerve injury model. The facial ...

Use of In Vivo Single-fiber Recording and Intact Dorsal Root Ganglion with Attached Sciatic Nerve to Examine the Mechanism of Conduction Failure

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for nerve fibers in the central and peripheral nervous systems. This method is particularly applicable to dorsal root ganglia (DRG), which are primary sensory neurons that exhibit a pseudo-unipolar structure of nervous processes. The patterns and features of the action potentials passed along axons are recordable in these neurons. The present study uses in vivo single-fiber recordings to observe the conduction failure of sciatic nerves in complete Freund&#8217;s adjuvant (CFA)-treated rats. As the underlying mechanism cannot be studied using in vivo single-fiber recordings, patch-clamp-recordings of DRG neurons are performed on preparations of intact DRG with the attached sciatic nerve. These recordings reveal a positive correlation between conduction failure and the rising slope of the after-hyperpolarization potential (AHP) of DRG neurons in CFA-treated animals. The protocol for in vivo single fiber-recordings allows the classification of nerve fibers via the measurement of conduction velocity and monitoring of abnormal conditions in nerve fibers in certain diseases. Intact DRG with attached peripheral nerve allows observation of the activity of DRG neurons in most physiological conditions. Conclusively, single-fiber recording combined with electrophysiological recording of intact DRGs is an effective method to examine the role of conduction failure during the analgesic process.

Single-fiber recording is an effective electrophysiological technique that is applicable to the central and peripheral nervous systems. Along with the preparation of intact DRG with the attached sciatic nerve, the mechanism of conduction failure is examined. Both protocols improve the understanding of the peripheral nervous system&#39;s relationship with pain.

Verwendung von In Vivo Single-Faser-Aufnahme und intakten DorsalWurzel Ganglion mit angeschlossenem Ischiasnerv, um den Mechanismus des Leitungsversagens zu untersuchen

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

3D Kinematische Analyse zur funktionellen Bewertung im Rattenmodell von Ischias-Nerven-Crush-Verletzungen

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...